Lipid bilayer coated mesoporous silica nanoparticles with a high loading capacity for one or more anticancer agents

ABSTRACT

A submicron structure comprising a silica body defining a plurality of pores that are suitable to receive molecules therein, and having a surface, and a phospholipid bilayer coating the surface, wherein said submicron structure has a maximum dimension of less than one micron, and wherein the phospholipid bilayer stably seals the plurality of pores; and wherein the submicron structure is a member of a monodisperse population of submicron structures.

This application claims the benefit of the filing date of U.S.provisional applications Ser. No. 61/773,013, filed Mar. 5, 2013 andSer. No. 61/858,388, filed Jul. 25, 2013, both of which are incorporatedby reference herein in their entireties.

This invention was made with Government support under Grant No. CA133697awarded by the National Institutes of Health. The Government has certainrights in the invention.

From the description herein, one skilled in the art can easily ascertainthe essential characteristics of this invention, and without departingfrom the spirit and scope thereof, can make changes and modifications ofthe invention to adapt it to various usage and conditions and to utilizethe present invention to its fullest extent. The following embodimentsare to be construed as merely illustrative, and not limiting of thescope of the invention in any way whatsoever. The entire disclosure ofall applications, patents, and publications cited herein are herebyincorporated by reference in their entirety, particularly with regard tothe subject matter for which they are cited.

BACKGROUND INFORMATION

Human pancreatic ductal adenocarcinoma (PDAC) is the fourth leadingcause of cancer-related death in the United States, with a mediansurvival period in PDAC patients of <6 months. While most cultured PDACcells are relatively sensitive to existing chemotherapeutic agents (e.g.Taxol, 5-FU, and gemcitabine), clinical treatments using free drug ordelivery by other means usually fails upon systemic administration.

While the availability of nanocarrier drug delivery systems is promisingfor cancer treatment due to protected drug encapsulation and targeteddelivery, this technology is still at an early stage from thetranslational medical perspective. One important obstacle is the low orlimited loading capacity that is often below the pharmaceuticalexpectation of a drug delivery carrier. This problem is exemplified inthe use of gemcitabine (GEM) that is widely used for treatment of humanpancreatic ductal adenocarcinoma (PDAC). GEM has a short half-life inblood stream and therefore its efficacy could be improved by thedevelopment of an improved carrier system. Current carriers, such asliposomes and certain submicron structures do not exhibit a sufficientloading capacity to deliver an adequately a cytotoxic dose of GEM atcancer site. For example, a GEM encapsulating liposome has been made bya procedure in which the free drug is added in the step of lipid filmrehydration. This conventional protocol usually leads to relatively lowdrug loading capacity (a yield of <8% (drug/liposome (w/w) drugloading).

There is a need for a carrier system with an improved loading capacityfor GEM or other agents that are useful for cancer treatment.Furthermore, there is a need for a carrier system into which can beloaded more than one such agent, particularly one agent that ishydrophobic and one which is hydrophilic.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows development of an efficient TGFβi carrier by supramolecularattachment to PEI-PEG coated MSNP. (A) A graphical representation of thePEI-PEG copolymer coated MSNP for supramolecular attachment of TGFβi,LY364947, via an H-bond mediated mechanism (see the insert box). TEMimage of TGFβi-MSNP was provided. (B) Measurement of the maximum loadingcapacity of LY364947 in PEI-PEG coated MSNP. Fixed amount of particle(i.e. 500 μg) was used for incubation with incremental amounts (50 to400 μg) of LY364947 at 25° Cover a 24 h time period. After thoroughlywashing in distilled water, the supernatants were separated bycentrifugation at 15,000 rpm for 30 min. The loading capacity wasquantitatively determined by using the LY364947 OD value of 269 nm (M5e,Molecular Device). Loading capacity (%, w/w)=[(Total minusnon-encapsulated weight of LY364947)/(weight of MSNP)]×100%. Particlesize and zeta potential in solution were measured by ZetaSizer Nano(Malvern Instruments Ltd., Worcestershire, UK) and provided in thebrackets. (C) Stability of TGFβi attachment in different solutions.TGFβi release was studied in deionized water, saline with 2% serum andDMEM supplemented with 10% FCS at different time points at 37° C.Release percentages were calculated by the following equation: Releasepercentage (%)=[(the weight of LY364947 in the supernatants)/(the totalweight of attached LY364947 at the starting point)]×100%. (D) TGFβirelease was studied in pH=5.5 aqueous solution for 24 h and comparedwith the release profile in PBS (pH=7.4).

FIG. 2 shows TGFβi-loaded MSNP disrupts PC interactions with EC invitro. (A) HDME cells (104 cells/mL) and HSM cells (5×103 cells/mL) werefirst stained by CellTracker™ Green CMFDA (Invitrogen, Grand Island,N.Y.) and CellTracker™ Red CMTPX (Invitrogen, Grand Island, N.Y.)according the manufacture's instruction 24 h before experiment. Afterlive cell staining, ECs were treated with 2 ng/mL of TGF-β for 3 h andPCs were treated with free TGF-β or TGFβi-MSNP at inhibitor dose of 1 μMfor 3 h. Subsequently, both cell types were co-cultured inMatrigel-coated 6-well plates for further incubation of 16 h at 37° C.PC/EC adhesions were quantitatively determined from five fields of threeindependent samples with the fluorescent microscope (Zeiss, Germany). *,P<0.05. (B) Use of immunofluorescence assay to determine the level ofSmad2 phosphorylation. PCs were seeded in 8-well chamber slides. 16 hpost cell seeding, PCs were treated with 2 ng/mL TGF-β for 3 h.Subsequently, the cells were treated with TGFβi-laden MSNP at theinhibitor dose of 1 μM for 1-24 h. For comparison, free TGFβi was usedto treat the cells at same dose. The treated cells were fixed,permeabilized, and incubated with primary antibody of anti-pSmad2 at 4°C. for 16 h. After PBS washing, the samples were further incubated withFITC-conjugated secondary antibody. The nucleus was stained by Hoechst33342. The slides were visualized under a fluorescence microscope. (C)The signal intensity of the green channel, reflecting activated Smad2(pSmad2), was calculated by Image J software (version 1.37c, NIH). *,P<0.05.

FIG. 3 shows TGFβi-loaded MSNP disrupts PC interactions with EC in BxPC3xenograft. (A) The tumor section was visualized by a Masson's trichromestaining, which showed a dense stroma including the heavy collagendeposition in blue in BxPC3 xenograft. “T” indicates tumor cells. Arrowspoint out stroma. (B) Tumor-bearing animals (tumor size of 0.8˜1.0 cm indiameter) were divided into two groups and received i.v. injection ofTGFβi-MSNP at inhibitor dose of 1 mg/kg (MSNP dose of 2 mg/kg). Salineand i.v. injection of free inhibitor at same dose were used as control.Tumor tissues were quickly collected 1-2 h post injection and OCTembedded for frozen section and dual color immunohistochemistrystaining. The procedure of the staining was provided in the methodsection. This dual color staining that labels EC maker CD31 in green(FITC) and PC maker NG2 in red (Alexa fluor 594) were used to comparethe effect of tumor blood vessel integrity and quantify the extent ofPC/EC association. Zoom-in images were provided at higher magnificationto show the extent of PC/EC association in each group. PC covered ECwere quantified in three random fields in each group. *, P<0.05.

FIG. 4 shows TEM ultrastructural analysis to elucidate the MSNP mediatedTGFβi delivery in BxPC3 xenograft. Electron microscopy to determine theultrastructure in BxPC3 tumor following administration to TGFβi-MSNP for2 h. RBC denotes red blood cell. Electron microscopy was powerful enoughto capture the porous structure of TGFβi-MSNP inside the tumor tissue,an ultrastructural feature that has not previously been accomplished.Additional TEM images are displayed in FIG. 9.

FIG. 5 shows TGFβi-loaded MSNP improves PDAC access of i.v. injected“hard” and “soft” nanoparticles in BxPC3 xenografts (A-B) An IVISoptical imaging system (Xenogen) was used to study the biodistributionof NIR dye labeled particles in the BxPC3 tumor-bearing mice. Tovisualize the luciferase expression in the cancer site, anesthetizedmice received intraperitoneal injection of 75 mg/kg d-Luciferin,followed 8-10 min later by obtaining the bioluminescence images.Reference fluorescence images were captured before treatment. Thetumor-bearing animals were first treated by i.v. injection of TGFβi-MSNP(inhibitor: 1 mg/kg) followed by i.v. injection of 50 mg/kg NIR-labeledPEI-PEG-MSNP or NIR-liposome with 1˜2 h interval. The in vivobiodistribution was compared with the mice received i.v. injection of 50mg/kg NIR dye labeled particles alone. Full panel of NIR images atdifferent time points were shown, e.g. in FIG. 11. The NIR fluorescentintensities were analyzed at different time points by software and shownin the lower panel of (A) and (B). *, P<0.05. (C-D) For PEI-PEG-MSNP, 60h after injection, the animals were sacrificed and tumor tissues as wellas major organs (heart, lung, spleen, liver, kidney, brain, and muscle)were collected for ex vivo imaging. ICP-OES was used to quantify the Siabundance in the major target organs using our established procedure. 28As a result of the shorter retention time of liposomes, we repeated theexperiments in (B) with a new batch of animals in which the tumor tissueand organs were harvested at 24 h for ex vivo imaging. The tumor tissuetogether with major organs were collected and used for ex vivo imaging.Around 100 mg of tumor, spleen, liver, and kidney was accurately weighedout, washed, and homogenated, and the fluorescence intensities perunitary amount of each organ were measured by a microplate reader (M5e,Molecular Devices). The biodistribution of each particle type wasexpressed as percent of total load of each nanoparticle distributing tothe individual organs. This percent is determined according to theformula [(particle quantity per unit mass tissue×tumor or organweight)/(total injected particle)]×100%. *, P<0.05, two-wave compared touse of particle alone.

FIG. 6 shows fluorescent images of tumor tissue sections to showTGFβi-MSNP improve the extent of liposome intratumoral distribution inBxPC3 xenografts. (A) In order to determine whether two-wave therapyalters the intratumoral biodistribution of i.v. injected texas redlabeled liposomes, these were i.v. injected into BXPC3 tumor-bearingmice in the absence or presence of prior TGFβi-MSNP injection. BXPC3tumor-bearing mice received intravenous administration of TGFβi-MSNP(inhibitor dose: 1 mg/kg; MSNP dose: 2 mg/kg) followed by red labeledliposome with 1-2 h interval.

Tumor tissues were collected 5 h post injection of the 2nd wave redlabeled liposome. Frozen histological sectioning of the OCT embeddedtumor tissues in each group was performed by the UCLA Division ofLaboratory Animal Medicine (DLAM) diagnostic laboratory services. Slideswere visualized under a fluorescence microscope (Zeiss, Germany). (B)Tumor tissue sections to show the tumor localization of the liposome inrelation to the ECs and PCs detected by CD31 and NG2 biomarkers usingimmunofluorescent staining. Part of tumor sections in each group wereincubated with a CD31 primary antibody and visualized by FITC-conjugatedsecondary antibody. The same section was further incubated with a NG2primary antibody and visualized by pacific blue-conjugated secondaryantibody. The red fluorescence of labeled liposome was also captured forthe same slide view, and merged images were prepared to showintratumoral distribution of the liposome in relation to the bloodvessels and their PCs coverage. High magnification images, labeled as“i”-“iii” in tumor received liposome alone and “iv”-“vi” in tumorreceived two-wave treatment, were provided.

FIG. 7 shows tumor growth inhibition and assessment of treatments onanimal weight and kidney histology in BxPC3-bearing nude mice. (A) Theanimal treatments are described in the method section. BxPC3 cells weresubcutaneously injected into mice 7 days before treatments (gray boxes).These animals received six i.v. injections (red boxes) every 3-6 days(green boxes) for 38 days as shown. The tumor inhibition effect oftwo-wave treatment was compared to saline, GEM-Lip alone, free GEM,empty liposome, and TGFβi-MSNP. Tumor size was accurately measured 1-2times per week. Tumor weight was calculated according to the formulaTumor weight (mg)=(length in mm)×(width in mm)2/2. *P<0.05, compared toGEM-Lip group. (B) The animal weights were recorded at indicate timepoints and expressed for the experimental duration. *P<0.05, compared tosaline; #P<0.05, compared to GEM-Lip. (C) Histological analyses ofkidney sections were performed by UCLA DLAM diagnostic laboratoryservices. The sections were stained with hematoxylin/eosin (H&E) andexamined by light microscopy. Representative images are shown. Highermagnification images were provided to shown the swelling and edema occurin Bowman's space, a morphological abnormality of GEM-induced renaltoxicity. White arrows point to normal Bowman's space in glomerulus, andthe black arrows point to the swelling and edema occur in Bowman'sspace.

FIG. 8 shows the display of a dysplastic stroma that includes PCcoverage of vascular fenestrations is an important consideration toimprove the tumor access of drug laden nanoparticle. Use of tumor tissuehistology to show that the PDAC tumor elicits a dense stromal barrier,which includes effective PC coverage of tumor blood vesselfenestrations, to the extent that blocks vascular access of i.v.injected red labeled liposome at BxPC3 tumor site. PCs were labeled inblue using a NG2 marker. ECs were labeled in green using a CD31 marker.One can see that liposome successfully extravagated from tumorfenestration in absence of PCs, however, many liposomes were trapped intumor blood vessel in which the ECs were efficiently covered by PCs. ThePC barrier can be visually shown by the sandwich-like blue-red-greenarrangement (that indicates PCs-liposomes-ECs) in the tumorfenestration. To improve liposome access at tumor fenestration, we havedesigned a two-wave treatment strategy with the intention of removingthe this effective PC coverage during the 1st wave therapy using TGFβiloaded MSNP, followed by a 2rd wave therapy in which a high dose ofcancer drug GEM is delivered by liposome. A scheme, on the right handside, was provided to conceptualize this two-wave approach for PDACtreatment.

FIG. 9 shows an electron microscopic image of a BxPC3 tumor section froman animal injected i.v. with TGFβi-MSNP. The images taken at differentresolutions show that the drug carrier (arrows) could be observed asintact, mono-disperse particles in the tumor blood vessel.High-resolution TEM images could resolve the porous structure of theMSNP. RBC: red blood cell. C: Collagen.

FIG. 10 shows optimization of GEM loading in the liposome platform bycreating a trans-liposomal-membrane ammonium sulfate gradient. (A) Upperpanel: A scheme to show the major steps in the (NH4)2SO4 mediated GEMloading. Lower panel: By adjusting the parameters during liposomesynthesis and drug loading, a highly efficient loading protocol wasestablished for each indicated formulation. The results showed that theDPPC:Cholesterol:DSPE-PEG2K (7:2:1) liposome (formulation #5) can beused for GEM loading using a (NH4)2SO4 mediated loading approach. Inorder to obtain high drug loading capacity, a list of parameters [(I)liposome formulation, (II) loading approach, (III) salt concentration,(IV) extent of salt removal, (V) extent of loading under variousnon-encapsulated GEM concentration, (VI) incubation temperature and(VII) incubation time] were systemically explored to develop the optimalloading conditions. (C) CyroEM image shows the primary size andunilamellar structure of GEM-laden liposome in saline. The thickness oflipid bilayer was determined to be 9 nm based on a quantitative analysisusing Image J software. (D) A hydrodynamic particle size and potentialin saline were measured. The synthesis optimization yielded a unilamilarliposome nanoparticle with a slightly negative zeta potential,hydrodynamic diameter of 137 nm in saline of a DPI index of 0.004. (E)Left: Cellular uptake of red-labeled liposomes in BxPC3 cells. Confocalmicroscopy was used to study the cellular uptake of liposome in BxPC3cells. Cells were exposed to 25 μg/mL labeled particles for 6 h. Cellnuclear were stained by Hochest dye. After cell membrane staining with 5μg/mL green-fluorescent wheat germ agglutinin (WGA 488), cells werevisualized using a confocal 1P/FCS inverted microscope. Right: MTS assaywas conducted for the GEM-loaded liposome delivered to these cells atincremental GEM doses over a 48 h period in BxPC3 cells. No cytotoxicitywas found using empty liposome (not shown). The controls were cellstreated with PBS or empty particles. The experiment was reproduced twotimes. (F) Demonstration of protective effect of liposomal encapsulationon CDA-mediated GEM inactivation.

FIG. 11 shows a full panel of NIR mages to cover all the time points inmice injected with 2nd wave NIR-MSNP as shown in FIG. 5A.

FIG. 12 shows the biodistribution of i.v. injected MR dye-labeled 50 nmamine-modified and PEGylated MSNP to the BxPC3-luc tumor xenograft modelin nude mice with or without TGFβi-MSNP.

FIG. 13 shows a full panel of NIR images to show the biodistribution of2nd wave NIR-liposomes at all time points for the experiment describedFIG. 5B.

FIG. 14 shows cryoEM images of lipid coated MSNP.

FIG. 15 shows confocal microscope images of Panc1 cells before and attime points after treatment with drug-loaded MSNP.

FIG. 16 shows results of time- and dose-dependent treatment of Panc1cells with paclitaxel-laden lipid coated MSNP on expression levels ofcytidine deaminase.

DESCRIPTION

The present invention relates, e.g., to a submicron structure whichexhibits a surprisingly large loading capacity for a variety ofsubstances, including small molecules, siRNAs and miRNAs. The submicronstructure comprise a silica body defining a plurality of pores that aresuitable to receive molecules therein, and having a surface, and aphospholipid bilayer coating the surface, wherein said submicronstructure has a maximum dimension of less than one micron (e.g. betweenabout 20 nm and about 300 nm, or between about 50 nm and about 200 nm).This submicron structure is sometimes referred to herein as a “submicronstructure of the invention” or as a “mesoporous silica nanoparticle(MSNP).”

The submicron structure can include a silica body defining a pluralityof pores that are suitable to receive molecules therein, and having asurface; and a phospholipid bilayer coating the surface; where thesubmicron structure has a maximum dimension of less than one micron, andwhere the phospholipid bilayer stably seals the plurality of pores; andwherein the submicron structure is a member of a monodisperse population(of submicron structures).

In embodiments of the invention, the submicron structure furthercomprises, loaded therein (bound to, encapsulated in, loaded with, intoor onto, laden with) an effective amount of at least one of thefollowing categories of therapeutic agents: a) a drug; b) an agent whichstabilizes the drug of a) against metabolic degradation; c) an agentwhich facilitates the delivery of the drug of a) to a target cell,tissue or organ; d) an agent which acts synergistically with the drug ofa); or e) one or more additional therapeutic agents, including, forexample. nucleic acids (e.g., siRNA or miRNA). In embodiments of theinvention, two or more of these categories of therapeutic agents areloaded together into the submicron structure.

For example, the submicron structure can be laden with both theanticancer drug Gemcytabine (GEM) and an agent which leads to inhibitionof its degradation, paclitaxel. The two agents act synergistically. Oneadvantage of the submicron particles of the present invention is thatthey can be loaded, as in this case, with both a hydrophilic molecule(GEM) and a hydrophobic molecule (paclitaxel).

In other embodiments of the invention, the submicron structures are usedin a multiwave (e.g. a two wave) method to treat a disease or condition,such as a cancer. For example, in some cancers, such as human pancreaticductal adenocarcinoma (PDAC), the tumor elicits a dense stromal barrierwhich includes effective pericyte coverage of tumor blood vesselfenestrations and blocks vascular access of cancer drug ladennanoparticles at the tumor site. In order to combat this blockage, in afirst wave, a submicron structure is attached to an inhibitory agentthat inhibits blockage or coverage of some or all of the tumor vascularfenestra and removes this pericyte coating. A submicron structure of theinvention is administered to a subject to be treated. For example, theagent can be an inhibitor of TGF-β kinase, which is part of the pathwayresponsible for pericyte adherence to the tumor vascular cells.

One typical such inhibitor is LY-3649747 (which not only is a potentinhibitor of the type 1 TGF-β receptor, but whose nitrogen display can,in embodiments of the invention, be used to attachpolyethyleneimine/polyethylene glycol (PEI/PEG) copolymer coated MSNPthrough H-bonding). Other suitable inhibitors of the TGF-β signalpathway include, e.g., SB505124, LY580276, LY550410, and LY364947. In asecond wave, an antitumor agent, such as a conventional chemotherapeuticdrug, siRNA, or miRNA is administered to the subject, either in a freeform, or in a liposome (such as the liposome described herein which hasa surprisingly high loading capacity) or in a nanoparticle (such as thesubmicron structure described herein which is coated with PEI-PEG, orthe submicron structure described herein which is coated with aphospholipid bilayer).

In embodiments of the invention, liposomes which are used in the secondwave of administration exhibit one or more of the following properties:mono-dispersed unilamellar colloidal vesicles of 100 nm;DPPC:Cholesterol:PEI-PEG=7:2:1; liposomes capable of forming homogenous˜100 nm carriers; ammonium sulphate mediated GEM loading; GEM loadingcapacity of about 20%; optimal loading by 120 nM (NH₄)₂SO₄, 3 cycles ofdialysis (6 mL against 1000 mL, 6 hours/cycle), use of 1 mg/mL free GEM,and incubation for 10 hours at 68° C.; stable storage for weeks.

In one embodiment of the invention, the agents for the first and secondwave are packaged together in the same submicron structure, which iscoated with a phospholipid bilayer. In embodiments of the invention, athird or further waves with additional chemotherapeutic agents, isadministered, in which each wave addresses sequential barriers to cancertreatment, so as to achieve an outcome that cannot be achieved byconventional chemotherapy or nanocarriers. The agents for each wave canbe delivered independently, or two or more of them can be packaged in asingle submicron structure of the invention.

Other advantages of the submicron structures coated with a phospholipidbilayer include monodisperse particle size distribution, which canfacilitate uniform cellular uptake of the particles; and control overthe dose(s) and ratio(s) of agents delivered together in the submicronstructure.

One aspect of the invention is a submicron structure including a silicabody defining a plurality of pores that are suitable to receivemolecules therein, and having a surface, and a phospholipid bilayercoating the surface, wherein said submicron structure has a maximumdimension of less than one micron, and wherein the phospholipid bilayerstably seals the plurality of pores; and wherein the submicron structureis a member of a monodisperse population. The term ‘monodispersepopulation’ refers to a plurality of particles (e.g., submicronstructures) in a colloidal system in which the suspended plurality ofparticles have substantially identical size and shape. For the purposesof the present invention, a monodisperse population can exhibit adeviation in diameter of 10% rms or less, or 5% rms or less.

The phospholipid bilayer can stably seal the plurality of pores. Inother words, submicron structures can retain molecules within the poresfor extended periods of time without substantial losses. In someembodiments, molecules can be retained within the submicron structuresfor 1, 2, 3, 4, 5, 6, or 7 days or more without substantial losses; orfor 1 week, 2 weeks, 3 weeks, or 4 weeks or more without substantiallosses; or for 1 month, 2 months, 3 months, 4 months, 5 months, or 6months or more without substantial losses. “Without substantial losses”can refer to a loss of 10% or less; 5% or less; or 2% or less ofmolecules retained within the pores.

A submicron particle can include about 5% w/w or greater of molecules(for example, therapeutic agents) within the pores; about 10% w/w orgreater; about 20% w/w or greater; about 30% w/w or greater; or about40% w/w or greater. The weight percent of molecules retained within thepores can be referred to as the loading capacity of submicronstructures.

Silica Body

The submicron structure includes a silica body that defines a pluralityof pores therein. For example, the silica body can be a mesoporoussilica nanoparticle. The fact that we refer to the body as a silica bodydoes not preclude materials other than silica from also beingincorporated within the silica body. In some embodiments, the silicabody may be substantially spherical with a plurality of pore openingsthrough the surface providing access to the pores. However, the silicabody can have shapes other than substantially spherical shapes in otherembodiments of the current invention. Generally, the silica body definesan outer surface between the pore openings, as well as side walls withinthe pores. The pores can extend through the silica body to another poreopening, or can extend only partially through the silica body such thatit has a bottom surface of the pore defined by the silica body.

In some embodiments, the silica body is mesoporous. In otherembodiments, the silica body is microporous. As used herein,“mesoporous” means having pores with a diameter between 2 nm and 50 nm,while “microporous” means having pores with a diameter smaller than 2nm. In general, the pores may be of any size, but in some embodimentsare large enough to contain one or more therapeutic compounds therein.In such embodiments, the pores allow small molecules, for example,therapeutic compound such as anticancer compounds to adhere or bind tothe inside surface of the pores, and to be released from the silica bodywhen used for therapeutic purposes. In some embodiments, the pores aresubstantially cylindrical.

Some embodiments of the invention include nanoparticles having porediameters between about 1 nm and about 10 nm in diameter. Otherembodiments include nanoparticles having pore diameters between about 1nm and about 5 nm. Other embodiments include particles having porediameters less than 2.5 nm. In other embodiments, the pore diameters arebetween 1.5 and 2.5 nm.

Silica nanoparticles having other pore sizes may be prepared, forexample, by using different surfactants or swelling agents during thepreparation of the silica nanoparticles.

The submicron structures according to some embodiments of the currentinvention may be referred to as nanoparticles. The term nanoparticles asused herein is intended the include particles as large as about 1000 nm.In general, particles larger than 300 nm may be less effective inentering living cells. In some embodiments, colloidal suspensions may beformed using a plurality of submicron structures according to someembodiments of the invention. In that case, larger particles can tend tosettle rather than remaining suspended in Brownian motion. As usedherein, size of the submicron structure refers to the size of theprimary particles, as measured by transmission electron microscopy (TEM)or similar visualization technique. Particle size does not refer toagglomerates in solution or suspension. Some embodiments includenanoparticles having an average maximum dimension between about 50 nmand about 1000 nm. Other embodiments include nanoparticles having anaverage maximum dimension between about 50 nm and about 500 nm. Otherembodiments include nanoparticles having an average maximum dimensionbetween about 50 nm and about 200 nm. In some embodiments, the averagemaximum dimension is greater than about 20 nm, greater than about 30 nm,greater than 40 nm, or greater than about 50 nm. Other embodimentsinclude nanoparticles having an average maximum dimension less thanabout 500 nm, less than about 300 nm, less than about 200 nm, less thanabout 100 nm or less than about 75 nm.

In some embodiments, the surface of the submicron structure ornanoparticle is unmodified. As used herein, an “unmodified” nanoparticlehas had no other functional groups added to the surface after formationof the nanoparticle. Unmodified nanoparticles have an anionic charge dueto free silyl hydroxide moieties present on the surface.

In embodiments of the invention, the submicron structure furthercomprises at least one of a valve assembly, a removable stopper assemblyor an impeller attached to the submicron structure's proximate or morepores. The submicron structure may comprise at least one of gold orsuper-paramagnetic core. A variety of submicron structures, and methodsof making them, are described in, for example, U.S. Patent ApplicationNos. 2010-0255103, 2010-0284924, 2010-0310465, 2012-0021034,2013-0046274, and 2012-0207795, each of which is incorporated byreference in its entirety.

Another aspect of the invention is a composition comprising a pluralityof submicron structures of the invention, wherein the submicronstructures are monodisperse with regard to size and uniformity.

Another aspect of the invention is a method of making a submicronstructure of the invention.

In a method, a silica body is prepared according to a sol-gel process(see, for example, Xia et al., ACS Nano, vol. 3, pp. 3273-3286, 2009;Jie et al., Small, vol. 3, pp. 1341-1346, 2007; each of which isincorporated by reference in its entirety). Subsequently, the pores ofthe silica body are loaded with molecules (e.g., a therapeutic agent). Aphospholipid bilayer is then formed on the surface of the silica body,thereby coating the surface. The phospholipid bilayer can stably sealthe molecules within the pores of the silica body. Because the moleculesare stably sealed within the pores, the submicron structures can have ahigh loading capacity for the molecules, and the high loading can bestably maintained prior to delivery (e.g., administration to a subject).

Forming the phospholipid bilayer can include contacting a suspension ofsilica bodies (e.g., pre-loaded silica bodies) with a solution ofphospholipids in a suitable solvent. The combined mixture can besupplied with energy (e.g., via sonication) to facilitate coating of thesilica body surface with a phospholipid bilayer. Numerous phospholipidssuitable for forming bilayers are known, including, but not limited to,1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The composition of thelipid bilayer can be adjusted as desired.

In the method, it is not required to pre-form phospholipid liposomesthat are contacted with the silica bodies; rather, a preformed film ofphospholipids is contacted with the silica bodies. This can avoid theneed to carry out a lipid phase exchange, which can complicate theprocess of forming the submicron structures.

Additional molecules (e.g., therapeutic agents) can be included in thelipid mixture used to form the lipid bilayer coating the silica body. Inone embodiment, paclitaxel can be included in the lipid mixture. Thus insome embodiments, the submicron structure can include two or moredifferent molecules, at least one of which is within the pores of thesilica body, and at least one of which is associated with thephospholipid bilayer.

In another aspect of the invention, the submicron structure furthercomprises one or more therapeutic agents. As used herein, a “therapeuticagent” is an agent that, by itself or in conjunction with one or moreother therapeutic agents, elicits a measurable amount of a therapeuticeffect (e.g., amelioration of a symptom) when administered to a subject.

One category of therapeutic agents that can be administered is aconventional drug, or anti-cancer agent, such as, e.g., GEM, taxol,doxorubicin, camptothecin, 5-FU, cisplatin, carboplatin or an siRNA ormiRNA designed and made by conventional methods to target a nucleic acidwhich encodes a protein that mediates a cancer.

Another category of therapeutic agents is an agent which stabilizes thedrug as noted above, e.g, against metabolic degradation. In addition toadministering paclitaxel in order to stabilize GEM, one can administer,e.g., agents which modulate oxidative stress, such a redox cyclingchemicals. Other small molecules or siRNAs or miRNAs that target a drugdegradation enzyme, such as CDA, can also be used.

Another category of therapeutic agents is an agent which facilitates thedelivery of the drug to a target cell, tissue, organ or tumor. Forexample, as discussed above, in order to remove or reduce stromal orpericyte blockage of tumor vasculature, an inhibitor of the TGF-13pathway, such as inhibitors of the type 1 or type 2 TGF-β receptors andkinases involved in those pathways can be administered. Alternatively orin addition, any of a variety of well-known inhibitors of the TGF-βreceptors or post receptor signaling pathways or transcription factorscan be used.

Another category of therapeutic agents is an agent that actssynergistically with a drug. In addition to the combination ofpaclitaxel and GEM, other pairs of synergistic agents can beadministered. These include, e.g., siRNA and chemodrugs, (e.g.doxorubicin and Pgp siRNA); paclitaxel and Bcl-2-targeted siRNA;paclitaxel and VEGF siRNA; doxorubicin and Bcl2 siRNA; folfurinox (4drugcombination); irinotecan and floxouridine; irinotecan and cisplatin;cytarabine and daunorubicin; doxorubicin and docetaxel; 6-mercaptopurineand daunorubicin; quercetin and vincristine; doxorubicin andphosphatidylinositol-3 kinase inhibitor; gemcitabine and doxorubicin;doxorubicin and a Pgp inhibitor, such as verapamil; cysplatinin orcarboplatin plus an aromatase inhibitor; methotrexate and all-transretinoic acid; and others that will be evident to a skilled worker.

Other therapeutic agents that can be administered by a method of thepresent invention will be evident to a skilled worker.

A submicron structure (particle) of the invention can be “loaded” withone or more therapeutic agents in a variety of ways. For example,substances such as hydrophilic substances can be incorporated into thepores, e.g. the substance can be introduced into the silica body duringthe process of forming the silica body, or the substance can beintroduced after the silica body has formed. A substance such as ahydrophobic substance can be attached to the phospholipid bilayer whichcoats the silica particle. The pores can also be loaded by phaseexchange with one or a combination of hydrophobic drugs (e.g.paclitaxel), allowing additional hydrophobic drugs to be added to thelipid bilayer.

The “subject” can be any of a variety of animals, including mammals suchas domestic animals (pets), laboratory animals, farm animals and humans.In one embodiment, the subject is a human having a cancer. In someembodiments, the subject has a cancer with a heavy stroma and pericytecoverage such as, e.g., PDAC, prostate cancer or a glioblastoma. Inembodiments in which an inhibitor of the TFG-P pathway is delivered witha submicron structure of the invention, the subject can have a conditionin which TFG-13 plays an important role in disease pathogenesis, suchas, e.g., neocartilage formation, organ fibrosis and aberrant immuneresponse.

An “effective” amount of a therapeutic agent is an amount that canelicit a measurable amount of a therapeutic effect, such as reduction ofa symptom of a disease or condition.

Generally, a submicron structure of the invention is administered to asubject systemically. Suitable routes of administration include, forexample, intravenous, intra-arterial, intraperitoneal, intramuscular, orsubcutaneous administration.

EXAMPLES Example I A. Method for Developing a Liposome that Contains aHigh Loading Capacity for Gemcitabine; Use of a Two-WaveNanotherapeutics Approach to Overcome Stromal Resistance and EnhanceGemcitabine Delivery in a Human Pancreatic Cancer Xenograft Model

While the availability of nanocarrier drug delivery systems is promisingfor cancer treatment due to protected drug encapsulation and targeteddelivery, this technology is still at an early stage from thetranslational medical perspective. One important obstacle is the low orlimited loading capacity that is often below the pharmaceuticalexpectation of a drug delivery carrier. This problem is exemplified inthe use of gemcitabine (GEM) that is widely used for treatment of humanpancreatic ductal adenocarcinoma (PDAC). GEM has a short half-life inblood stream and therefore its efficacy could be improved by ananocarrier such as liposome, provided that the liposome of a sufficientloading capacity could deliver an adequately a cytotoxic dose of GEM atcancer site. A GEM encapsulating liposome has been made by a procedurein which the free drug is added in the step of lipid film rehydration.This conventional protocol usually leads to relatively low drug loadingcapacity (a yield of <8% (drug/liposome (w/w) drug loading).

The present inventors have found that by creating an ammonium sulfate((NH₄)₂SO₄) gradient inside the liposome, under optimal conditions, byan active exchange reaction, it is possible to develop an improved drugloading protocol capable of highly efficient GEM encapsulation thatgenerally results in about 20% loading capacity (drug/liposome, w/w).The salt gradient inside the liposome improves GEM loading and leads toa gel-like precipitate of GEM inside the liposome. Without wishing to bebound by any particular mechanism, it is suggested that this highloading occurs because the amphipathic GEM molecule can easily diffusethrough the liposome bilayer as un-protonated species, and issubsequently trapped inside the liposome due to a protonation reactionthat converts the amphipathic into hydrophilic molecules. The protonatedproducts of less diffusion ability can be stabilized as gel-like drugprecipitate (i.e. (GEM-NH₃)₂SO₄) inside the liposomes.

One method of the invention comprises a rehydration procedure usingammonium sulfate containing solution for loading for GEM. Shown hereinare analyses of each parameter during the synthesis and GEM loading,including liposome formulation, ammonium sulfate concentration, extentof salt removal, drug loading time, temperature, amount of free GEM,etc. Advantageously, the loading approach also leads to improved drugstability inside the liposome. Other agents that are structurallysimilar to GEM can also be efficiently encapsulated into liposomes by amethod of the present invention.

Due to the high loading ability including the potential of liposomemodification (i.e. PEG, active ligand, fluorescent labeling, etc), thisliposomal GEM delivery platform exhibits good cancer killing abilityboth in vitro and in vivo. Moreover, the GEM-laden liposome will be anideal “second wave” particle that can be used in the multi-wave PDACtherapy, as described elsewhere herein.

Aspects of this embodiment include the following:

1. A liposome comprising at least about 20% gemcitabine (GEM)/lipid(wt/wt), wherein the GEM is in the form of a gel-like precipitate (e.g.(GEM-NH₃)₂SO₄). In embodiments of the invention, the liposome is furthermodified (e.g. with PEG, an active ligand, fluorescent label, etc.).2. A method for making a liposome comprising about at least about 20%gemcitabine (GEM)/lipid (wt/wt), wherein the GEM is in the form agel-like precipitate (e.g. (GEM-NH₃)₂SO₄), the method comprising (a)hydrating a thin lipid film (e.g., using the formulation DPPC:Cholesterol: DSPE-PEG2K=7:2:1) in the presence of about 120 nM(N11₄)₂SO₄ to form a liposome comprising an ammonium sulfate gradient,then (b) loading GEM into the liposome (e.g. by an equilibrationmethod).

This method can comprise

-   -   preparing a lipid film,    -   hydrating the lipid film in a buffer comprising about 120 nM        (NH₄)₂SO₄ to form a liposome comprising an ammonium sulfate        gradient,    -   removing non-encapsulated salts from the liposome (e.g., by        about 3 dialysis cycles against about 1000 mL, for about 6        h/cycle), then    -   incubating the liposomes with free GEM (e.g. at a concentration        of about 1 mg/mL at about 60° C., for about 10 h), and    -   removing non-encapsulated GEM.        3. A method for introducing GEM into a cell (in vitro or in        vivo, e.g. from or in a subject having a cancer, such as        pancreatic cancer), comprising contacting the cell with a        liposome of claim 1, under conditions that are effective for        efficient delivery of the liposome into the cell. In one        embodiment of the invention, the method is for treating a tumor        in pancreatic cancer, such as human pancreatic ductal        adenocarcinoma (PDAC), wherein the method further comprises (a)        in a first wave, inhibiting the tumor TGF-β signaling pathway by        contacting the tumor with a TGF-β inhibitor laden mesoporous        silica nanoparticle (MSNP) capable of manipulating the human        pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment        by releasing a small molecule inhibitor, such as LY-364947        (C₁₇H₁₂N₄), thereby removing pericyte coverage from the tumor,        then (b) in a second wave, contacting the tumor with the        liposome comprising GEM.

Abstract

Pancreatic cancer elicits a dense stromal response in which pericytecoverage of tumor vasculature presents a barrier that interferes withliposomal delivery of gemcitabine. In order to improve liposomaldelivery, we used a mesoporous silica nanoparticle to deliver a smallmolecule inhibitor of the TGF-β pathway to decrease pericyte coverageand improve gemcitabine delivery to a human xenograft tumor. This dualwave approach provided effective tumor cell killing compared to freedrug or liposome-encapsulated drug, thereby demonstrating the utility ofan engineered approach to stromal drug resistance.

Introduction

Human pancreatic ductal adenocarcinoma (PDAC) is the fourth leadingcause of cancer-related death in the United States, with a mediansurvival of <6 months.¹ Since PDAC is typically diagnosed at a latestage, many PDAC cases cannot be considered for surgery because ofmetastases and local spread to the superior mesenteric blood vessels atthe time of diagnosis.^(2, 3) While chemotherapy is often the onlyoption, even this form of treatment is characterized by poor efficacyand severe side effects in PDAC patients. While most cultured PDAC cellsare relatively sensitive to chemotherapeutic agents such as gemcitabine(GEM), Taxol, and 5-FU, clinical treatment is often unsuccessful becauseof the dense stromal barrier, which is a histological hallmark of PDACas compared to other cancer diseases.⁴ The desmoplastic stroma iscomprised of a dense extracellular matrix, as well as a variety ofnon-cancerous cells, including the presence of pericytes that blocksvascular fenestrations and prevents vascular access of cancer drugs andother therapeutic agents .⁴⁻⁸ This includes interference in the deliveryof drug-laden nanocarriers in animal PDAC models.⁵⁻⁸ Pericyte coverageof more than 70% of the tumor vasculature significantly differentiatesPDAC from other cancer types that exhibit a less dense stroma, e.g.,glioblastoma or renal carcinoma in which the pericyte coverage islimited to 10-20% of the blood vessels.⁴⁻⁶ Mammary and colon carcinomafall somewhere in between ⁴⁻⁶ Thus, the development of efficacious andsafe chemotherapy for PDAC is a big challenge.

A number of strategies have been entertained to improve the efficacy ofdelivery of chemotherapy and decreasing drug side effects in PDAC. Theseefforts have included improvement of the pharmacokinetic profile,tumor-specific targeting and attempts to overcome the resistance of thestromal barrier.⁹⁻¹² One promising approach is to take advantage of theability of nanocarriers to encapsulate and deliver chemotherapeuticagents to improve the stability and cytotoxic killing of PDAC cells. Forinstance, free GEM, which is a first-line chemotherapeutic agent forPDAC, has a very short half-life in vivo and can be rapidly decomposedby a cytidine deaminase (CDA) degradation in the circulation and at thetumor site.¹³ Use of nano carrier, such as unilamellar pegylatedliposome, has been shown to increase GEM plasma half-life andintratumoral drug concentration to the extent that a 10 times lower drugdose could be used for tumor inhibition in mice, without signs oftoxicity.¹¹ An additional exciting advance with the introduction ofnanocarriers is the potential to target the stromal chemoresistancepathway that interferes in tumor vascular access. For penetration ofanticancer drugs, either in their free or nanocarrier format, is animportant factor limiting drug efficacy and bioavailability at the PDACsite.¹⁴ Tumor angiogenesis is controlled by a number of growth factorpathways, including the important role of the transforming growth factorbeta (TGF-β) pathway in promoting pericyte coverage.¹⁵ TGF-β stabilizescapillary-like structures during neo-angiogenesis and is alsoresponsible for the differentiation of mesenchymal cells into pericytes(PCs) that cover endothelial cells (ECs), leading to the formation ofintact blood vessels.^(16, 17) Thus, TGF-β signaling inhibition presentsone of the promising targets to affect change in the vascular access ofcancer drugs and nanocarriers to tumor sites.^(7, 18) Vascular accesscan also be improved by reducing the collagen content of the vasculatureand stroma throughout the tumor interstitium.¹⁹

The program is discussed above now allows us to propose an engineeredapproach to PDAC drug delivery through the use of nanocarriers thatprovide protected encapsulation as well as improving vascular accessthrough targeting of stromal elements. By combining these principles, wepropose a two-wave therapeutic procedure in which the first step is togain vascular access by a mesoporous silica nanoparticle (MSNP)nanocarrier that inhibits the TGF-β pathways by delivering a smallmolecule inhibitor (LY364947), also referred to as “TGFβi”, followed bysubsequent delivery of a liposome that has been optimized for efficientGEM encapsulation and delivery. In this communication, we provideproof-of-principle testing to demonstrate that it is possible to enhanceGEM delivery to a human pancreatic xenograft in a nude mice model. Wedemonstrate the development of a MSNP carrier that can be used forsupramolecular attachment of TGFβi, including the ability of thiscarrier to interfere in PCs recruitment to ECs in vitro and to a humanxenograft PDAC tumor in vivo. We demonstrate that this carrier caneffectively enhance vascular access of a 2^(nd) wave of GEM-loadedliposomes to the same tumor in vivo. We demonstrate that GEM loading inthis liposome can be dramatically increased by creating an ammoniumsulfate gradient inside the liposome which through GEM protonation couldincrease its transport from the incubation medium. We went on todemonstrate increased therapeutic efficacy and reduced side effects ofthis dual wave platform in relation to free GEM.

Results

Development of an Efficient TGFβi Carrier by Supramolecular Attachmentto MSNP

The highly coordinated action of various growth factors, includingheterotypic PCs interaction with ECs, leads to the formation andstabilization of tumor blood vessels.²⁰ In this complex regulation,TGF-β, a well-known vasculature modulator, regulates various processesleading to vessel maturation, inhibition of ECs proliferation andmigration, induction of PCs differentiation, and maintaining theintegrity of the microvasculature.²⁰⁻²² Use of medicinal chemicalsynthesis and screening, the extensive knowledge of TGF-β signaltransduction pathway has led to the development of a group of compoundsthat can interfere in signaling by the TGF-β receptor-I, such asSB505124, LY580276, LY550410, and LY364947. LY364947, a nitrogenheterocyclic compound (FIG. 1A), is a potent inhibitor of thereceptor-associated SMAD kinase in vitro and in vivo test.²³⁻²⁵ Theelectronegative nitrogen atoms in LY364947 introduce the opportunity touse supramolecular or electrostatic chemistry for attachment to afunctionalized nanocarrier surface. Such an opportunity presented itselffor our multifunctional MSNP platform that has previously been developedfor protected and efficient systemic delivery of a variety of cargoes tocancer cells and xenograft tumors in mice ²⁶⁻³² This includes thedevelopment of a 50 nm MSNP core that is coated with apolyethyleneimine-polyethylene glycol (PEI-PEG) copolymer, whichpresents free amine groups that could be H-bonded to the nitrogen groupon the inhibitor (FIG. 1A). Another advantage of this carrier is thatthe PEI-PEG coating is stably attached, provides monodispersion of MSNPin blood and decreases update by the reticuloendothelial system, so asto allow a long circulatory half-life and effective delivery of drugsand/or siRNA to breast and cervical tumor sites.^(28, 31) To assess thepotential effectiveness of the copolymer-coated MSNP to deliverLY364947, we used fixed amount of particle (i.e. 500 μg) for incubationwith incremental amounts (50 to 400 μg) of the kinase inhibitor at 25°C. over a 24 h time period. After thoroughly washing in distilled water,the loading capacity was quantitatively determined by using the LY364947 OD value of 269 nm. This demonstrated a maximum drug loadingcapacity of ˜74% (inhibitor/particle, w/w), which reflects the abundanceof H-binding donors and acceptors on PEI and the inhibitor, respectively(FIG. 1 B). Moreover, the soft structure of PEI facilitatesconformational changes that allow strong hydrogen bonding andincorporation of the drug on the particle surface (FIG. 1B). This leadsto a slight increase in the hydrodynamic particle size from 120 nm to130 nm at the maximum loading capacity for the inhibitor. While thenon-drug bonded MSNP exhibited a zeta potential value of +45 mV inwater, binding of the drug inhibitor decreased this value to +30 mV(FIG. 1B). FIG. 1C demonstrates that the drug-bound particles are stablysuspended in water, saline (plus 2% serum) and cell culture medium for72 h. A subsequent release study showed that the TGFβi could be releasedfrom the MSNP in a time-dependent manner by lowering of the pH of thesolution to 5.5 (FIG. 1D). Approximately 40% weight percentage TGFβicould be released within 24 h.

TGFβi-Loaded MSNP Disrupts PC Interactions with EC in Vitro and in Vivo

To investigate the effects of TGFβi on the co-migration of culturedhuman vascular smooth muscle cells (used as a surrogate PC) with humanmicrovascular EC, we used a Matrigel assay³³ to compare the effect ofTGFβi-loaded MSNP with the free inhibitor (FIG. 2). ECs and PCs werestained with CellTracker™ Green and CellTracker™ Red, respectively. FIG.2A demonstrates that the percentage of PC/EC association wassignificantly inhibited if the TGFβi was delivered by MSNP as comparedto the inhibitory effect of free inhibitor at 1 μM. Representativefluorescent images of the cellular co-migration are shown on the righthand side of the figure. Upon binding to type I/II TGF-β receptors, thegrowth factor induces the phosphorylation of the C-terminal SXS motif ofthe-associated kinases, Smad2 and Smad3 .³⁴ Looking at Smad2phosphorylation in PCs, we used an immunochemical technique thatdiscerns anti-pSmad2 by a secondary FITC-conjugated antibody under aconfocal microscope (FIG. 2B).³⁵ This demonstrated efficient andsustained inhibition of Smad2 phosphorylation for up to 24 h in PCstreated with TGFβi-MSNP compared to cells exposed to free inhibitor,which only suppressed pSmad2 for 6 h (FIG. 2B). Quantitative assessmentof the green fluorescence intensity by Image J software confirmed astatistically significant and sustained inhibition of Smad2phosphorylation by TGFβi-MSNP (FIG. 2C).

In order to determine whether TGFβi delivery to a PDAC tumor site willhave the same effect on PC co-localization with ECs in the tumor, weestablished BxPC3 xenografts in nude mice, because it has previouslybeen shown that this human PDAC model gives rise to an aberrant anddense infiltrative stroma in which tumor blood vessels are embedded.³⁶The presence of a dense stroma was confirmed by Masson's trichromestaining, which shows heavy collagen deposition in the BxPC3 xenograft(FIG. 3A). To achieve effective TGFβi delivery by our MSNP carrier werelied on its effective biodistribution properties and a relatively longcirculatory half-life as a result of limited RES uptake due to the PEGcoating.³⁷ TGFβi-MSNP was injected intravenously at inhibitor dose of 1mg/kg (equivalent to a MSNP dose of 2 mg/kg) in nude mice expressingtumors ranging from 0.8-1.0 cm in diameter. Tail vein injections ofsaline or the free inhibitor (at same dose) were used as controls. Todemonstrate the impact on PC/EC co-localization, dual-colorimmunohistochemistry was used for detecting CD31 staining in ECs with agreen fluorescent dye (FITC), and NG2 in PCs with a red fluorescencemarker (Alexa fluor 594) (FIG. 3).^(7, 33) These results showed thatTGFβi-MSNP injection could significantly disrupt the composite (yellow)fluorescence staining resulting from PC overlap with ECs (FIG. 3B).Little separation of the green and red fluorescence distribution wasseen in saline treated animals, while injection of the free inhibitorresulted in a slight but non-significant reduction of the compositefluorescence staining (FIG. 3B). The likelihood that TGFβi was beingdelivered to the tumor vascular bed is suggested by the ultrastructuralresolution of monodisperse mesoporous particles in small blood vessels,as recorded by electron microscopy (FIG. 4). More TEM images are shownbelow. In FIG. 9. Since the H-bonding of the drug to the polymer surfaceis pH sensitive, it is possible that acidification at the tumor site maycontribute to the release of TGFβi.³⁸ This could also explain theabsence of any other vascular abnormalities following TGFβi-MSNPinjection in other organs where PCs reside (such as the brain, whichwill be described later).

Collectively, above data provide proof-of-principal testing of TGFβibound MSNP as a potential nanocarrier that can be used to engineer PDACstromal barrier for the ease of nano drug delivery.

TGFβi-Loaded MSNP Improves PDAC Access of i.v. Injected “Hard” and“Soft” Nanoparticles in BxPC3 Xenografts

Since PCs regulate capillary blood flow as well as vascular access, thenext question became whether TGFβi-MSNP could improve the egress ofnanocarriers at the BxPC3 xenograft site.³⁹ We tested this possibilitythrough the use of “hard” (100 nm PEI-PEG coated MSNP) and “soft” (130nm liposome) nanocarriers in an imaginable biodistribution experiment innude mice. These 2^(nd) wave particles were designed with near-infrared(NIR) tags to provide high photon penetration in animal tissues, asdescribed previously by us.^(28, 31) TEM or cryoEM images of theparticles are provided in FIGS. 5A and 5B. Detailed characterization isprovided in FIG. 11. To visualize the tumor growth in mice, BxPC-3 cellswere stably transfected with a luciferase vector and used for obtainingbioluminescence images in the mice following intraperitoneal (i.p.)injection of d-Luciferin (FIGS. 5A and 5B, first row). Initial referenceimages showed very low NIR background in the tumor-bearing animals(FIGS. 5A and 5B, second row). Subsequently, the tumor-bearing animalswere i.v. injected with TGFβi-MSNP (containing 1 mg/kg of theinhibitor), followed after 1-2 h interval, by i.v. injection of 50 mg/kgMR-labeled MSNPs or liposomes. This biodistribution was compared to micereceiving i.v. injection of 50 mg/kg NIR-labeled particles in theabsence of prior treatment with TGFβi-MSNP. NIR fluorescence images werecaptured at different time points as shown in the 3^(rd) and 4^(th) rowsin FIGS. 5A and 5B. The full panel of NIR images appears in FIG. 11. Inthe absence of prior TGFβi-MSNP treatment, the images indicate that thelabeled MSNPs were rapidly taken up in the spleen and kidney within 24 h(FIG. 5A, first column) While PEI-PEG coated MSNP has been sequentiallyoptimized for systematic administration and passive retention incervical and breast cancer xenografts^(28, 31), there was limited egressin stroma-rich BxPC3 xenografts in nude mice (FIG. 5A, first column)While we still observed particle retention in the RES of mice injectedwith TGFβi-MSNP, these animals showed prominent particle retention atthe xenograft sites by 24 h, suggesting a strong particle retentioneffect (FIG. 5A, second column) Following the software analysis of theMR fluorescence intensities at different time points as shown in thelower panel of FIGS. 5A, prior TGFβi-MSNP administration resulted in asignificant increase in the fluorescence intensity by 40 h, whereuponthe signal was sustained for at least 60 h. Very little change influorescence intensity was observed in the tumor tissue receivingNIR-MSNP alone. Similar enhanced retention of a 50 nm amine-modified,PEGylated MSNP at the xenograft site was observed following 1^(st) waveTGFβi-MSNP administration as shown in FIG. 12. In parallel experiments,the effect of TGFβi-MSNP was also studied to visualize the retention ofa liposomal particle (DPPC: Cholesterol: DSPE-PEG=7:2:1) at the BxPC3xenograft site. To develop a NIR-labeled liposome, Dylight 680-DMPE(<0.1%, w/w) was incorporated into the lipid mixture. Compared to thebiodistribution of the i.v. injected liposome alone (FIG. 5B, firstcolumn), there was a significant increase in fluorescence intensity attumor site in the mice that were injected with TGFβi-MSNP (FIG. 5B,second column) Interestingly, the liposome accumulated with more rapidkinetics than the MSNP and could be observed in the xenograft 1 h posti.v. injection. The images also demonstrate that the liposomesdisappeared faster than the silica nanoparticles, suggesting that theliposomes are rapidly metabolized in vivo (FIG. 5B and FIG. 13). Similarto MSNPs, semiquantitative imaging analysis showed that TGFβi-MSNPsignificantly increases liposome retention at the tumor site compared toinjecting the liposomes alone (lower panel of FIG. 5B).

The mice receiving the NIR-labeled MSNPs were sacrificed at 60 h postinjection, and ex vivo fluorescence images were obtained for the tumortissue as well as major organs (FIG. 5C, upper panel). Consistent withthe live animal imaging results, prior TGFβi-MSNP treatment wasassociated with increased fluorescence intensity in tumor tissuecompared to animals receiving the 2^(nd) wave treatment alone. Bothanimal groups showed abundant particle distribution to the liver,spleen, lung, and the kidney. Following ex vivo imaging, the collectedorgans were weighed and used for Si elemental analysis by inductivelycoupled plasma optical emission spectrometry (ICP-OES). This allowedquantitative analysis of the particle distribution, expressed as apercentage (%) of the total mass of administered particles. While ˜7% ofthe particles could be seen to biodistribute to the tumor tissue at 60 hin animals treated with TGFβi-MSNP, it is at least 10 times higher thanthe particle content of the BxPC3 tumor site in animals receiving theNIR-MSNP alone (FIG. 5C, lower panel). As a result of the shorterretention time of liposomes, we repeated the experiments in FIG. 5B witha new batch of animals in which the tumor tissue and organs wereharvested at 24 h for ex vivo imaging. Consistent with the live imagingresults, prior treatment with TGFβi-MSNP significantly increasedfluorescence intensity at the tumor tissue compared to animals injectedwith liposomes alone. Both groups showed abundant distribution to liver,spleen, lung and kidney (FIG. 5D, upper panel). Calculation offluorescence intensity using our established protocol,³¹ prior treatmentwith TGFβi-MSNP resulted in the retention of ˜7% of the administeredliposomes as compared to ˜1.8% of the injected dose in control tumors.This amount, as shown in the use of two-wave treatment approach, isapproximately 4 folds higher than the animals injected with liposomesalone (FIG. 5D, lower panel).

TGFβi-MSNP Improve the Extent of Liposome Intratumoral Distribution inBxPC3 Xenografts

In order to determine whether two-wave therapy alters the intratumoralbiodistribution of texas red labeled liposomes, these were i.v. injectedinto BxPC3 tumor-bearing mice in the absence or presence of priorTGFβi-MSNP injection. Visual inspection of the fluorescence distributionunder low magnification demonstrated a heterogeneous intratumoraldistribution if the liposomes were injected alone (FIG. 6A, left upperpanel). Most of the fluorescence intensity localized in the tumorperimeter. High magnification imaging further demonstrated that thelabeled liposomes could be visualized as fluorescent intracellular dotsthat appear in the cytosol and perinuclear regions (FIG. 6A, left lowerpanel). This is in keeping with the cellular internalization ofliposomes, some of which could be taken up in acidifying endosomalcompartments in the tumor cells.¹⁰ In contrast, there was a dramaticchange in the intratumoral distribution of the liposomes following the1^(st) wave delivery of TGFβi-MSNP (FIG. 6A, right upper panel).Additional immunohistochemical staining for CD31 with a FITC-conjugatedantibody and NG2 with a pacific blue-conjugated antibody allowed us todetermine liposomal localization in relation to ECs and PCs,respectively (FIG. 6B). As compared to single wave delivery of liposomesalone, two-wave treatment resulted in more abundant and homogenousliposome distribution in the xenograft tissue. Moreover, merging of blueand green fluorescent images demonstrated the disassociation in ECs andPCs during two-wave treatment (FIG. 6B, regions “vi”, “v”, and “vi”) ascompared to the co-localization of these cells (FIG. 6B, regions “i”,“ii”, and “iii”) in animals injected with liposomes only. Allconsidered, these data demonstrate that TGFβi-MSNP pre-treatment allowsvascular access and widespread intratumoral distribution of engineerednanoparticles. This prompted the question of whether two-wave therapycan be used to improve the efficacy of GEM-laden liposomes in PDACtumor-bearing mice.

Two-Wave Treatment Improves the Efficacy of Gemcitabine Treatment ofBxPC3 Tumors

To demonstrate the possible effect of TGFβi-MSNP for treatment efficacyof BxPC3 xenografts, we decided to use the same liposomal carrierdepicted in FIG. 5B for encapsulate the delivery of GEM. In order toimprove its loading capacity, we created an (NH₄)₂SO₄ salt gradient inthe liposome,⁴⁰ which through protonation of the drug could increase GEMtransport from the incubation medium as a function of ammonium sulfateconcentration, extent of salt removal, the effect of temperature, drugloading time, and amount of free GEM, etc (FIG. 10).^(11, 40-42) Thisallowed us to achieve GEM loading capacity of 19.8% w/w, which stands incontrast to a conventional loading in which showed a loading capacity of10.3% w/w. Full details about the liposome design, detailedphysicochemical characterization, optimization of drug loading,stability, cellular uptake, and ability to protect the drug against CDAinactivation are described in FIG. 10.

In the animal efficacy experiment, xenograft-bearing nude mice were 1.v. injected with 101 mg/kg of the liposomes (GEM dose: 20 mg/kg) 1-2 hafter the i.v. injection of TGFβi-MSNP (TGFβi dose of 1 mg/kg), every3-6 days for 38 days (FIG. 7A). The controls included animals injectedwith saline, free GEM, empty liposomes, TGFβi-MSNP alone, andGEM-liposomes alone. Since our previous studies have shown that emptyMSNP lacks anticancer activity,^(28, 31) we did not include this as anegative control in our animal experiments. When comparing the effect ontumor size, the GEM liposome showed a significantly higher rate of tumorshrinkage than the free drug (FIG. 7A). The use of two-wave treatmentbeyond 25 days, demonstrated an additional and significantly higher rateof tumor inhibition compared to the use of the GEM-liposome alone. Thisdelay in observing the effect of prior TGFβi-MSNP treatment could be dueto the effect of tumor stage, with the stromal effects and vascularaccess becoming a problem beyond 25 days. No tumor inhibition was foundwith saline treatment, TGFβi-MSNP alone or the use of empty liposomes(FIG. 7A).

Two-Wave Therapy Reduces the Systemic Toxicity of GEM

The safety of nanocarrier delivery system is of key importance in theassessment of this therapeutic platform. This includes the inherentsafety of the carrier as well as the possible benefits that may accruedue to drug encapsulation. Safety assessment was performed by monitoringtotal body weight, blood chemistry, and histological examination ofmajor organs. Compared to saline-treated

BxPC3 tumor-bearing mice, no significant body weight changes wereobserved during the administration of empty liposomes, GEM-liposomes, orTGFβi-MSNP plus GEM-liposomes. In contrast, animals receiving free GEMadministration showed a reduced weight gain (FIG. 7B). While none of theanimals showed a significant elevation of biomarkers that denote majororgan dysfunction, free GEM resulted in intermediate nephrotoxicity,⁴³which manifested as glomerular swelling and edema of Bowman's space inthe kidney glomerulus. However, this histological change did not occurin other groups and histological examination of the liver and spleen didnot show any gross pathology in any of the experimental groups.

Discussion

In this study, we used an engineered approach wherein TGFβi-MSNPtreatment was used to initially target the tumor stroma to decrease PCcoverage of EC, followed by the delivery of GEM-laden liposomes thatwere effectively distributed throughout the tumor tissue, resulting inenhanced killing of the cancer cells after a window of 25 days followingtreatment. In order to achieve optimal in vivo efficacy, both particlewaves were optimally designed to prolong circulation time in the blood,reduce RES uptake, and carry an effective drug payload to the cancersite. Thus, the co-polymer coated MSNP could deliver a high load of aTGFβi, which was supramolecularly attached to PEI, and through slowrelease could interfere in PCs adherence to the tumor vasculature at thexenograft site. This allowed nanocarrier egress through the vascularfenestrations, with the ability to enhance encapsulated GEM delivery tothe tumor site. The 2^(nd) wave of delivery was achieved by an optimallydesigned liposome characterized by PEG surface display as well as theability to import and retain the protonated GEM at a ˜20% w/w loadingcapacity. Release of the encapsulated GEM throughout the xenograft tumorwas associated with increased cancer cells and less renal toxicitycompared to the free drug. All considered, these data demonstrate thattwo-wave nanotherapeutics can be used to target the effect of the stromain PDAC drug delivery, while also providing protected delivery of GEM tothe tumor site. This allows further testing of this platform inorthotopic human pancreatic cancer models in immunocompromised animalsas well as consideration for phase I human trials based on the two-wavetreatment concept.

Utilizing our multifunctional MSNP platform to conductproof-of-principle studies in various human cancer models, we haveobserved a wide range of challenges imposed by micro-heterogeneity inthe tumor environment that goes beyond the concept of an enhancedpermeability and retention effect. While undoubtedly vascularabnormalities such as large fenestrations could contribute tonanocarrier egress at the cancer site, there are a number oftumor-specific biological impediments to vascular access. In the case ofPDAC, the display of a dysplastic stroma that includes PC coverage ofvascular fenestrations is an important consideration (see FIG. 8). Thus,it is important to consider this impediment in the design ofnanocarriers for drug delivery, including the consideration of anengineered approach towards specific barriers, which can be targeted byindependent waves of therapy that ultimately provide effective ofkilling and elimination of the cancer tissue. However, while severalcreative ideas have emerged to attempt multistagenanotherapeutics^(44, 45) or combination therapy^(7, 14, 46, 47) withthe view to improve systemic drug delivery through increased bloodvessel permeability, tumor penetration or reducing the effect of druginactivation enzymes, most of the research efforts concentrated oncancer cell killing with few efforts being directed to the cancermicroenvironment.³¹ In fact, the tumor microenvironment is a verycomplex system that differs from the normal tissue environment andsignificantly influences the efficacy of nano delivery systems. Thisincludes the contribution from a variety of components in the tumormicroenvironment, i.e., biophysicochemical factors (hypoxia, acidosis,high interstitial fluid pressure), heterogeneous cellular componentsother than cancer cells (endothelial cells and pericytes,cancer-associated fibroblasts, and inflammatory cells), and non-cellularcomponents (extracellular matrix, matrix metalloproteinase, solublegrowth factors and their receptors, and integrins).³¹ Thus, one or moreof these heterogeneous components could be the target(s) of anengineered approach. One example is the use of macrolide-modified goldnanorods that were designed to target and activate antitumor potentialof macrophages.⁴⁸ This research demonstrated that the nanorodspreferentially accumulate in tumor-associated macrophages, leading to asignificantly enhanced killing potential of breast cancer cells.⁴⁸ Theimportance of manipulating the tumor microenvironment is furtherillustrated by our study in which the dense stromal barrier could beimpacted by delivery of a small molecule inhibitor, thereby enhancingencapsulated drug delivery by a 2^(nd) wave nanocarrier. While we havedemonstrated similar, but lesser stromal effects in a drug resistanthuman breast cancer xenograft, the accompanying microvascularheterogeneity was still responsible for preventing total disappearanceof the tumor.³¹

For PDAC, this problem is much more accentuated, thereby differentiatingthis from cancer types with a less prominent stroma, therefore allowinga prominent EPR effect. ^(7, 49)

The idea of targeting the PDAC stroma in clinical studies has beenaddressed by using of PEGylated hyaluronidase PH20 (PEGPH20), whichtargets hyaluronan, a tumor matrix component, which is responsible for ahigh interstitial fluid pressure (IFP) that interfere in penetration.⁵⁰Results from an ongoing clinical trial has demonstrated that thecombination of GEM with PEGPH20 treatment can improve the stromalbarrier, allowing chemotherapeutic agents drugs to freely permeate thecancer site.^(51, 52) This differs from our animal study in which we didnot observe a significant change in the collagen content, probably dueto the relatively short duration of treatment. In a recent phase IIIclinical trial in previously untreated PDAC patients with metastaticdisease, it has been demonstrated that the combination of Abraxane^(R)(paclitaxel/albumin complex) with GEM could induce a statisticallysignificant improvement in overall survival compared to patientsreceiving drug alone (median of 8.5 vs. 6.7 months).⁵³ In the animalstudy, it was demonstrated that paclitaxel is capable of defeating thedesmoplastic stroma, and increasing the GEM content 2.8-fold in thetumor as a result of the reducing CDA enzyme levels.^(46, 54) Therefore,it will be interesting to test in future whether combined delivery ofGEM and paclitaxel by a nanocarrier could be used as an even moreeffective 2nd wave of treatment.

The TGF-β superfamily plays an important role in cancer biology.²⁴ Thisincludes a role in tumor neo-angiogenesis in which the interaction ofPCs with ECs play a role in formation of intact blood vessels.¹⁷ Theeffects of inhibiting the TGF-β signaling pathway has been demonstratedin multiple in vitro and in vivo models, i.e. tumor xenograft models, aretinal vascular model, and a 3D PC/EC co-culture model.^(7, 18, 22, 55)Collectively, these studies indicate that TGF-β maintains the integrityand function of the microvasculature while interference in this pathwayoften leads to dissociation of EC from PC and impaired EC barrierfunction.^(7, 18, 22, 55) Our results also confirm a previous studyshowing that low dose intraperitoneal injection of TGFβi promotesvascular access and accumulation of nanoparticles and macromolecules inBxPC3 subcutaneous xenograft model and OCUM-2MLN orthotopic gastriccancer model^(7,18) In addition, TGF-βnegatively regulates local tumorimmune responses and one can envisage that TGFβi-MSNP may promote thefunction of tumor antigen specific CD8⁺ T cells in the localimmunosuppressive tumor microenvironment.^(56, 576)

Finally, we want to address the loading capacity of drugs in thenanocarrier. Good drug loading is important for efficacious cancer cellkilling as well as the potential to decrease systemic toxicity bylowering the amount of the nanomaterial that needs to be injected. Wewere able to optimize the effects of the LY364947 delivering particle byusing supramolecular attachment of its electronegative nitrogen residuesto the hydrogen atoms in PEI, with the ability to achieve a 74% (w/w)loading capacity. Not only have we been able to achieve a nanocarrierthat can be used for cancer applications, as demonstrated in thiscommunication, but also for the treatment of tissue inflammation,pulmonary fibrosis and arthritis. In the case of a liposomal carrier, ahigh loading capacity (˜20%) for GEM was achieved by creating anammonium sulfate gradient in the liposome. This allowed intra-liposomalretention of the drug, which is protonated after diffusion through theliposomal membrane.⁴⁰ It has also been shown that the encapsulated GEMis stabilized as a gel-like precipitate inside the liposome (FIG. 10).⁴⁰

In conclusion, by addressing a specific aspect of the biology of PDAC,we could develop an engineered approach in a human xenograft modelwherein we could improve vascular access past the stromal barrier aswell as delivery of chemotherapeutic agent. We propose that thisapproach is much more rational than the conventional passive and activedelivery approach of chemotherapeutic agents by nanocarriers. However,we do not exclude the possibility that the addition of targeting ligandsto our nanocarriers could further enhance their efficacy.

Studies are carried out with the submicron structures described inExample II (drug(s)-laden lipid bilayer coated MSNP), using the sameprocedures described in this Example I. For example, near infraredlabeled particles are synthesized for in vivo biodistribution studies intumor xenograft bearing nude mice, and in vivo efficacy tests arecarried out. It is expected that the in vivo efficacy can be at least aseffective as for the subunit structures described in present Example I.

Materials and Methods Materials and Experimental Details

The materials and experimental methods are described in detail elsewhereherein.

Synthesis of PEI-PEG Coated Mesoporous Silica Nanoparticles and NIRLabeling.

The synthesis of the 50 nm MSNP core was carried out as previouslydescribed by us, using a sol-gel chemistry procedure.^(28, 31) Theparticle surface was further modified using electrostatic attachment ofa 1.8 kD PEI polymer, which was subsequently used for covalentattachment of 5 kD PEG. To perform PEI coating, 10 mg of MSNP wassuspended in 1 mL of 2.5 mg/mL PEI 1.8 kD ethanolic solution. Thesolution was sonicated and stirred for 30 min. The particles werefurther washed in ethanol to remove excess PEI and trace amount ofsurfactant. The PEI-coated particle was subsequently transferred into1.5 mL of DMF, mixed with 50 mg of activated poly(ethylene glycol)methyl ether (m-PEG, MW 5 kD), and stirred for 24 h. The nanoparticleswere washed with DMF and ethanol and resuspended in water.^(28, 31) TheNIR fluorescent dye DyLight 680 NHS ester was used for particlelabeling. 10 mg particles were suspended in 1 mL of DMF and mixed with0.1 mg of Dylight 680. The reaction took place under an inert atmosphereduring stirring at room temperature for 12 h. The particles werecentrifuged and washed with deionized water.²⁸

Assessment of TGFβi Loading Capacity and Binding Stability

Various volumes (10 μL, 20 μL, 40 μL, 80 μL) of 5 mg/mL LY364947 DMSOsolutions were suspended in 1 mL of 0.5 mg/mL MSNP aqueous suspension.The mixture solutions were stirred at 25° C. for 24 h, and washed 3times with deionized water. After centrifugation at 15,000 rpm for 30min, the supernatants were collected to obtain OD value of LY364947 at269 nm (M5e, Molecular Devices, USA). The loading capacity wascalculated as follows: Loading capacity (%, w/w) =[(Total minusnon-encapsulated weight of LY364947)/(weight of MSNPs)]×100%. In orderto determine the stability of LY364947 attachment, the drug release wasstudied in deionized water, saline containing 2% fetal calf serum orDMEM supplemented with 10% FCS for time periods ranging from 0-72 h at37° C. Following samples centrifugation at 15,000 rpm for 30 min, therelease percentage was calculated according to the following equation:Release percentage (%)=[(the weight of LY364947 in thesupernatants)/(the total weight of attached LY364947 at the startingpoint)]×100%.

Cell Lines and Cell Culture

Human microvascular endothelial cell (HDME, used as ECs model) waspurchased from ScienCell Research Laboratories (Carlsbad, Calif.). TheECs were cultured in endothelial cell medium (ECM, Carlsbad, Calif.)containing 5% FBS, 1% endothelial cell growth supplement (ScienCell,Carlsbad, Calif.), 100 U/mL penicillin, 100 pg/mL streptomycin. Humansmooth muscle (HSM, a pericyte-like cell type and used as PCs model³³)was purchased from American Type Culture Collection (ATCC). The PCs werecultured in ATCC-formulated F-12K medium containing 0.05 mg/mL ascorbicacid, 0.01 mg/mL insulin, 0.01 mg/mL transferring, 10 ng/mL sodiumselenite, 0.03 mg/mL endothelial cell growth supplement, 10 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid(TES), and 10% FBS. BxPC-3 cells were purchased from ATCC and culturedin Dulbecco's modified eagle medium (DMEM) (Carlsbad, Calif.) containing10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mML-glutamine.

Matrigel Assay

To study the effect of TGFβi-MSNP on PC/EC interaction, the Matrigelassay was performed using a modified method in the literature.³³ Inorder to distinguish the PCs and ECs in the Matrigel assay, HDME cells(10⁴ cells/mL) and HSM cells (5×10³ cells/mL) were first stained byCellTracker™ Green CMFDA (Invitrogen, Grand Island, N.Y.) andCellTracker™ Red CMTPX (Invitrogen, Grand Island, N.Y.) according themanufacture's instruction 24 h before experiment. After live cellstaining, ECs were treated with 2 ng/mL of TGF-β for 3 h and PCs weretreated with free TGF-β or TGFβi-MSNP at inhibitor dose of 1 μM for 3 h.Subsequently, both cell types were co-cultured in Matrigel-coated 6-wellplates for further incubation of 16 h at 37° C. PC/EC adhesions werequantitatively determined from five fields of three independent sampleswith the fluorescent microscope (Zeiss, Germany).

Smad2 Activation Assay

Smad2 activation was determined using an immunofluorescent staining in8-well chamber slides in which 4×10⁴ PCs were cultured in each wellcontaining 0.4 mL culture medium. 16 h post cell seeding, PCs weretreated with 2 ng/mL TGF-β for 3 h. Subsequently, the cells were treatedwith TGFβ-laden MSNP at the inhibitor dose of 1 μM for 1-24 h. Forcomparison, free TGFβi was used to treat the cells at identical dose.Subsequently, PCs cells grown on chamber slides were fixed,permeabilized, and stained for pSmad2 with a standardimmunocytochemistry protocol. pSmad2 staining was performed by using a1:500 dilution of primary anti-pSmad2 antibody (Abcam, Cambridge, Mass.)for 16 h at 4° C. This was followed by a 1:500 diluted FITC-conjugatedsecondary antibody (Santa Cruz, USA) for 1 h at room temperature. Thenuclei were stained with Hoechst 33342. Slides were visualized under aconfocal microscope (Leica Confocal 1P/FCS). The signal intensity ofgreen channel, revealing activated Smad2, was calculated by Image Jsoftware (version 1.37c, NIH).

Establishment of BxPC3 Tumor Xenograft Model

Athymic BALB/c nu/nu female mice (6 weeks) were purchased from theCharles River Laboratory and maintained under pathogen-free conditions.All animal experiments were performed using protocols approved by theUCLA Animal Research Committee. For tumor visualization in mice usingoptical imaging, permanent luciferase transfection using lentivirus wasperformed in BxPC3 cells. To grow tumor xenograft, BxPC3-luc cellsuspension (0.1 mL, 5×10⁶ cells/mL) was injected subcutaneously intonude mice. For efficacy experiment, the mice were used for varioustreatments 7 days post tumor implantation. To perform imagingexperiment, the tumor bearing animals were used 3-4 weeks after tumorimplantation of the tumor size of 0.8-1 cm in diameter.

Bio-distribution

In order to determine the effect of the 1^(st) wave particle onimproving the distribution of systemically administrated 2^(rd)nanoparticle in BxPC3 tumor, the imaging studies were performed. Tovisualize the tumor growth, BxPC3-luc were used for obtainingbioluminescence images in the mice following intraperitoneal (i.p.)injection of D-Luciferin at 75 mg/kg. Eight to ten minutes afterinjection, bioluminescence images were acquired using an IVIS ImagingSystem (Xenogen, Toronto, ON, Canada). The mice were first intravenouslyadministrated with TGFβi-laden MSNP at an inhibitor dose of 1 mg/kg,which is equivalent to a MSNP dose of 2 mg/kg. To visualize the 2^(rd)wave particle in vivo, the NIR-MSNP or NIR-liposome were used. One totwo hour after TGFβi-laden MSNP injection, the mice were intravenouslyadministrated with 50 mg/kg of MR dye-labeled particles. Thefluorescence images were taken at indicated time points. This treatmentwas compared to the mice received i.v. injection of NIR dye-labeled MSNPor liposome alone at 50 mg/kg. The tumor tissue together with majororgans (heart, lung, spleen, liver, kidney, brain and cardiac muscle)were collected and used for ex vivo image.

Transmission Electron Microscopy of BxPC3 Tumor Received TGFβ-Laden MSNPTreatment

BxPC3 tumor-bearing mice (tumor size: 0.8-1 cm in diameter) wereintravenously treated with TGFβi-laden MSNP at inhibitor dose of 1 mg/kg(MSNP dose: 2 mg/kg). The tumor biopsies were rapidly collected 2 h postinjection, washed in PBS and immediately fixed with 2.5% glutaraldehydein PBS at room temperature for 2 h and stored at 4° C. for overnight.Further sample preparation and sectioning were performed by ElectronMicroscopy Services Center in Brain Research Institute at UCLA. Briefly,after secondary fixation in 1% O_(s)O₄ in PBS, the samples weredehydrated in a graded ethanol series, treated with propylene oxide, andembedded in resin. Approximately 60-70 nm thick sections were cut on aLeica ultramicrotome and picked up on Formvar-coated copper grids. Thesections were examined in a CM120 electron microscope (Philips).

Co-Staining of the Markers of ECs and PCs in Tumor Section

The tumor tissues were rapidly embedded by OCT reagent before sectioningto provide 4 μm thick slices. The slices were washed three times in PBSand fixed. For ECs staining, the sections were first incubated withrat-anti-mouse CD31 monoclonal antibody (1:500) at 4° C. overnight.After removal of the primary antibody and washing in PBS for threetimes, FITC-conjugated goat-anti-rat IgG (1:500) was added and incubatedat room temperature for 1 h. For PCs staining, the same section wasfurther incubated with primary antibody of NG2 (1:500) at 4° C.overnight and followed by Alexa594- or pacific blue-conjugated secondaryantibody (1:1000) at room temperature for 1 h. All the incubations wereperformed in dark. The slides were visualized under a fluorescencemicroscope. The PC coverage was counted from three randomly selectedfields.

Nude Mouse Studies to Determine the Efficacy of Two-Wave Treatment onTumor Shrinkage.

One week after tumor implantation, the BxPC-3 tumor-bearing mice wererandomly divided into six groups. These groups were used for comparingthe effects of saline, free liposome, TGFβi-MSNP alone, free GEM,GEM-Lip alone, and two-wave treatment, respectively. Each animal intwo-wave group received i.v. injection of TGFβi-MSNP at inhibitor doseof 1 mg/kg (MSNP dose: 2 mg/kg) followed by a liposome dose of 101 mg/kg(GEM dose: 20 mg/kg) with 1-2 h interval, during each injection, 6injections in a 38 days time period (FIG. 6A). The free GEM and GEMloaded liposome groups received the same drug dose in the absence ofTGFβi-MSNP pre-treatment. The groups treated with saline, emptyliposome, and TGFβi-MSNP, were used as control. The body weight andtumor size were accurately recorded one to two times per week. Tumorweight was calculated according to the formula Tumor weight (mg)=(lengthin mm)×(width in mm)²/2.

References for Example IA

1. Hezel, A. F.; Kimmelman, A. C.; Stanger, B. Z.; Bardeesy, N.;DePinho, R. A.,

Genetics and biology of pancreatic ductal adenocarcinoma. Genes &Development 2006, 20, (10), 1218-1249.

2. Wray, C. J.; Ahmad, S. A.; Matthews, J. B.; Lowy, A. M., Surgery forPancreatic Cancer: Recent Controversies and Current Practice.Gastroenterology 2005, 128, (6), 1626-1641.

3. Kleeff, J.; Reiser, C.; Hinz, U.; Bachmann, J.; Debus, J.; Jaeger,D.; Friess, H.; Büchler, M. W., Surgery for Recurrent Pancreatic DuctalAdenocarcinoma. Annals of Surgery 2007, 245, 566-572.

4. Erkan, M.; Hausmann, S.; Michalski, C. W.; Fingerle, A. A.; Dobritz,M.; Kleeff, J.; Friess, H., The role of stroma in pancreatic cancer:diagnostic and therapeutic implications. Nat Rev Gastroenterol Hepatol2012, 9, (8), 454-467.

5. Morikawa, S.; Baluk, P.; Kaidoh, T.; Haskell, A.; Jain, R. K.;McDonald, D. M., Abnormalities in Pericytes on Blood Vessels andEndothelial Sprouts in Tumors. The American journal of pathology 2002,160, (3), 985-1000.

6. Armulik, A.; Abramsson, A.; Betsholtz, C., Endothelial/PericyteInteractions. Circulation Research 2005, 97, (6), 512-523.

7. Kano, M. R.; Bae, Y.; Iwata, C.; Morishita, Y.; Yashiro, M.; Oka, M.;Fujii, T.; Komuro, A.; Kiyono, K.; Kaminishi, M.; Hirakawa, K.; Ouchi,Y.; Nishiyama, N.; Kataoka, K.; Miyazono, K., Improvement ofcancer-targeting therapy, using nanocarriers for intractable solidtumors by inhibition of TGF-β signaling. Proceedings of the NationalAcademy of Sciences 2007, 104, (9), 3460-3465.

8. Zhang, L.; Nishihara, H.; Kano, M. R., Pericyte-Coverage of HumanTumor Vasculature and Nanoparticle Permeability. Biological andPharmaceutical Bulletin 2012, 35, (5), 761-766.

9. Patra, C. R.; Bhattacharya, R.; Wang, E.; Katarya, A.; Lau, J. S.;Dutta, S.; Muders, M.; Wang, S.; Buhrow, S. A.; Safgren, S. L.;Yaszemski, M. J.; Reid, J. M.; Ames, M. M.; Mukherjee, P.; Mukhopadhyay,D., Targeted Delivery of Gemcitabine to Pancreatic Adenocarcinoma UsingCetuximab as a Targeting Agent. Cancer Research 2008, 68, (6),1970-1978.

10. Straubinger, R. M.; Hong, K.; Friend, D. S.; Papahadjopoulos, D.,Endocytosis of liposomes and intracellular fate of encapsulatedmolecules: Encounter with a low pH compartment after internalization incoated vesicles. Cell 1983, 32, (4), 1069-1079.

11. Celano, M.; Calvagno, M.; Bulotta, S.; Paolino, D.; Arturi, F.;Rotiroti, D.; Filetti, S.; Fresta, M.; Russo, D., Cytotoxic effects ofGemcitabine-loaded liposomes in human anaplastic thyroid carcinomacells. BMC Cancer 2004, 4, (1), 63.

12. Erkan, M., Understanding the stroma of pancreatic cancer:co-evolution of the microenvironment with epithelial carcinogenesis. TheJournal of Pathology 2013, n/a-n/a.

13. Baker, J. A. R.; Wickremsinhe, E. R.; Li, C. H.; Oluyedun, O. A.;Dantzig, A. H.; Hall, S. D.; Qian, Y.-w.; Ring, B. J.; Wrighton, S. A.;Guo, Y., Pharmacogenomics of Gemcitabine Metabolism: Functional Analysisof Genetic Variants in Cytidine Deaminase and Deoxycytidine Kinase. DrugMetabolism and Disposition 2013, 41, (3), 541-545.

14. Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.;Agemy, L.; Greenwald, D. R.; Ruoslahti, E., Coadministration of aTumor-Penetrating Peptide Enhances the Efficacy of Cancer Drugs. Science2010, 328, (5981), 1031-1035.

15. Goel, S.; Duda, D. G.; Xu, L.; Munn, L. L.; Boucher, Y.; Fukumura,D.; Jain, R. K.,

Normalization of the Vasculature for Treatment of Cancer and OtherDiseases. Physiological Reviews 2011, 91, (3), 1071-1121.

16. Winkler, E. A.; Bell, R. D.; Zlokovic, B. V., Central nervous systempericytes in health and disease. Nat Neurosci 2011, 14, (11), 1398-1405.

17. Darland, D. C.; D'Amore, P. A., TGFβ is required for the formationof capillary-like structures in three-dimensional cocultures of 10T1/2and endothelial cells. Angiogenesis 2001, 4, (1), 11-20.

18. CabralH; MatsumotoY; MizunoK; ChenQ; MurakamiM; KimuraM; TeradaY;Kano, M. R.; MiyazonoK; UesakaM; NishiyamaN; KataokaK, Accumulation ofsub-100 nm polymeric micelles in poorly permeable tumours depends onsize. Nat Nano 2011, 6, (12), 815-823.

19. Lammerts, E.; Roswall, P.; Sundberg, C.; Gotwals, P. J.;Koteliansky, V. E.; Reed, R. K.; Heldin, N.-E.; Rubin, K., Interferencewith TGF-β1 and -β3 in tumor stroma lowers tumor interstitial fluidpressure independently of growth in experimental carcinoma.International Journal of Cancer 2002, 102, (5), 453-462.

20. ten Dijke, P.; Arthur, H. M., Extracellular control of TGF[beta]signalling in vascular development and disease. Nat Rev Mol Cell Biol2007, 8, (11), 857-869.

21. Antonelli-Orlidge, A.; Saunders, K. B.; Smith, S. R.; D'Amore, P.A., An activated form of transforming growth factor beta is produced bycocultures of endothelial cells and pericytes. Proceedings of theNational Academy of Sciences 1989, 86, (12), 4544-4548.

22. Walshe, T. E.; Saint-Geniez, M.; Maharaj, A. S. R.; Sekiyama, E.;Maldonado, A. E.; D'Amore, P. A., TGF-beta Is Required for VascularBarrier Function, Endothelial Survival and Homeostasis of the AdultMicrovasculature. PLoS ONE 2009, 4, (4), e5149.

23. Kano, M. R.; Komuta, Y.; Iwata, C.; Oka, M.; Shirai, Y.-t.;Morishita, Y.; Ouchi, Y.; Kataoka, K.; Miyazono, K., Comparison of theeffects of the kinase inhibitors imatinib, sorafenib, and transforminggrowth factor-β receptor inhibitor on extravasation of nanoparticlesfrom neovasculature. Cancer Science 2009, 100, (1), 173-180.

24. Yingling, J. M.; Blanchard, K. L.; Sawyer, J. S., Development ofTGF-[beta] signalling inhibitors for cancer therapy. Nat Rev Drug Discov2004, 3, (12), 1011-1022.

25. Vogt, J.; Traynor, R.; Sapkota, G. P., The specificities of smallmolecule inhibitors of the TGF-beta and BMP pathways. CellularSignalling 2011, 23, (11), 1831-1842.

26. Meng, H.; Liong, M.; Xia, T.; Li, Z.; Ji, Z.; Zink, J. I.; Nel, A.E., Engineered Design of Mesoporous Silica Nanoparticles to DeliverDoxorubicin and P-Glycoprotein siRNA to Overcome Drug Resistance in aCancer Cell Line. ACS Nano 2010, 4, (8), 4539-4550.

27. Meng, H.; Xue, M.; Xia, T.; Zhao, Y.-L.; Tamanoi, F.; Stoddart, J.F.; Zink, J. I.; Nel, A. E., Autonomous in Vitro Anticancer Drug Releasefrom Mesoporous Silica Nanoparticles by pH-Sensitive Nanovalves. Journalof the American Chemical Society 2010, 132 12690-12697.

28. Meng, H.; Xue, M.; Xia, T.; Ji, Z.; Tarn, D. Y.; Zink, J. I.; Nel,A. E., Use of Size and a Copolymer Design Feature To Improve theBiodistribution and the Enhanced Permeability and Retention Effect ofDoxorubicin-Loaded Mesoporous Silica Nanoparticles in a Murine XenograftTumor Model. ACS Nano 2011, 5, 4131-4144.

29. Meng, H.; Yang, S.; Li, Z.; Xia, T.; Chen, J.; Ji, Z.; Zhang, H.;Wang, X.; Lin, S.; Huang, C.; Zhou, Z. H.; Zink, J. I.; Nel, A. E.,Aspect Ratio Determines the Quantity of Mesoporous Silica NanoparticleUptake by a Small GTPase-Dependent Macropinocytosis Mechanism. ACS Nano2011, 5 4434-4447.

30. Meng, H.; Xue, M.; Zink, J. I.; Nel, A. E., Development ofPharmaceutically Adapted Mesoporous Silica Nanoparticles Platform. TheJournal of Physical Chemistry Letters 2012, 3, (3), 358-359.

31. Meng, H.; Mai, W. X.; Zhang, H.; Xue, M.; Xia, T.; Lin, S.; Wang,X.; Zhao, Y.; Ji, Z.; Zink, J. I.; Nel, A. E., Codelivery of an OptimalDrug/siRNA Combination Using Mesoporous Silica Nanoparticles To OvercomeDrug Resistance in Breast Cancer in Vitro and in Vivo. ACS Nano 2013, 7,(2), 994-1005.

32. Mai, W. X.; Meng, H., Mesoporous silica nanoparticles: Amultifunctional nano therapeutic system. Integrative Biology 2013, 5,(1), 19-28.

33. Song, N.; Huang, Y.; Shi, H.; Yuan, S.; Ding, Y.; Song, X.; Fu, Y.;Luo, Y., Overexpression of Platelet-Derived Growth Factor-BB IncreasesTumor Pericyte Content via Stromal-Derived Factor-1 j/CXCR4 Axis. CancerResearch 2009, 69, (15), 6057-6064.

34. Varelas, X.; Samavarchi-Tehrani, P.; Narimatsu, M.; Weiss, A.;Cockburn, K.; Larsen, B. G.; Rossant, J.; Wrana, J. L., The CrumbsComplex Couples Cell Density Sensing to Hippo-Dependent Control of theTGF-β-SMAD Pathway. Developmental Cell 2010, 19, (6), 831-844.

35. Chen, J.; Shearer, G. C.; Chen, Q.; Healy, C. L.; Beyer, A. J.;Nareddy, V. B.; Gerdes, A. M.; Harris, W. S.; O'Connell, T. D.; Wang,D., Omega-3 Fatty Acids Prevent Pressure Overload-Induced CardiacFibrosis Through Activation of Cyclic GMP/Protein Kinase G Signaling inCardiac Fibroblasts. Circulation 2011, 123, (6), 584-593.

36. Farace, P.; Merigo, F.; Fiorini, S.; Nicolato, E.; Tambalo, S.;Daducci, A.; Degrassi, A.; Sbarbati, A.; Rubello, D.; Marzola, P.,DCE-MRI using small-molecular and albumin-binding contrast agents inexperimental carcinomas with different stromal content. European Journalof Radiology 2011, 78, (1), 52-59.

37. Ferrari, M., Cancer nanotechnology: opportunities and challenges.Nat Rev Cancer 2005, 5, (3), 161-171.

38. Estrella, V.; Chen, T.; Lloyd, M.; Wojtkowiak, J.; Cornnell, H. H.;Ibrahim-Hashim, A.; Bailey, K.; Balagurunathan, Y.; Rothberg, J. M.;Sloane, B. F.; Johnson, J.; Gatenby, R. A.; Gillies, R. J., AcidityGenerated by the Tumor Microenvironment Drives Local Invasion. CancerResearch 2013, 73, (5), 1524-1535.

39. Fakhrejahani, E.; Toi, M., Tumor Angiogenesis: Pericytes andMaturation Are Not to Be Ignored.Journal of Oncology 2012, Article ID261750.

40. Federico, C.; Morittu, V.; Britti, D.; Trapasso, E.; Cosco, D.,Gemcitabine-loaded liposomes: rationale, potentialities and futureperspectives. International Journal of Nanomedicine 2012, 2012,5423-5436.

41. Haran, G.; Cohen, R.; Bar, L. K.; Barenholz, Y., Transmembraneammonium sulfate gradients in liposomes produce efficient and stableentrapment of amphipathic weak bases. Biochimica et Biophysica Acta(BBA)—Biomembranes 1993, 1151, (2), 201-215.

42. Castelli, F.; Raudino, A.; Fresta, M., A mechanistic study of thepermeation kinetics through biomembrane models: Gemcitabine-phospholipidbilayer interaction. Journal of Colloid and Interface Science 2005, 285,(1), 110-117.

43. Glezerman, I.; Kris, M.; Miller, V.; Seshan, S.; Flombaum, C.,Gemcitabine nephrotoxicity and hemolytic uremic syndrome: report of 29cases from a single institution. Clin Nephrol 2009, 71, 130-139.

44. von Maltzahn, G.; Park, J.-H.; Lin, K. Y.; Singh, N.; Schw??ppe, C.;Mesters, R.; Berdel, W. E.; Ruoslahti, E.; Sailor, M. J.; Bhatia, S. N.,Nanoparticles that communicate in vivo to amplify tumour targeting. NatMater 2011, 10, (7), 545-552.

45. Watson, K. D.; Lai, C.-Y.; Qin, S.; Kruse, D. E.; Lin, Y.-C.; Seo,J. W.; Cardiff, R. D.; Mahakian, L. M.; Beegle, J.; Ingham, E. S.;Curry, F.-R.; Reed, R. K.; Ferrara, K. W., Ultrasound IncreasesNanoparticle Delivery by Reducing Intratumoral Pressure and IncreasingTransport in Epithelial and Epithelial-Mesenchymal Transition Tumors.Cancer Research 2012, 72, (6), 1485-1493.

46. Frese, K. K.; Neesse, A.; Cook, N.; Bapiro, T. E.; Lolkema, M. P.;Jodrell, D. I.; Tuveson, D. A., nab-Paclitaxel Potentiates GemcitabineActivity by Reducing Cytidine Deaminase Levels in a Mouse Model ofPancreatic Cancer. Cancer Discovery 2012, 2, (3), 260-269.

47. Liu, J.; Liao, S.; Diop-Frimpong, B.; Chen, W.; God, S.; Naxerova,K.; Ancukiewicz, M.; Boucher, Y.; Jain, R. K.; Xu, L., TGF-β blockadeimproves the distribution and efficacy of therapeutics in breastcarcinoma by normalizing the tumor stroma. Proceedings of the NationalAcademy of Sciences 2012, 109, (41), 16618-16623.

48. Dreaden, E. C.; Mwakwari, S. C.; Austin, L. A.; Kieffer, M. J.;Oyelere, A. K.; El-Sayed, M. A., Small Molecule-Gold Nanorod ConjugatesSelectively Target and Induce Macrophage Cytotoxicity towards BreastCancer Cells. Small 2012, 8, (18), 2819-2822.

49. Fang, J.; Nakamura, H.; Maeda, H., The EPR effect: Unique featuresof tumor blood vessels for drug delivery, factors involved, andlimitations and augmentation of the effect. Advanced Drug DeliveryReviews 2011, 63, (3), 136-151.

50. Jacobetz, M. A.; Chan, D. S.; Neesse, A.; Bapiro, T. E.; Cook, N.;Frese, K. K.; Feig, C.; Nakagawa, T.; Caldwell, M. E.; Zecchini, H. I.;Lolkema, M. P.; Jiang, P.; Kultti, A.; Thompson, C. B.; Maneval, D. C.;Jodrell, D. I.; Frost, G. I.; Shepard, H. M.; Skepper, J. N.; Tuveson,D. A., Hyaluronan impairs vascular function and drug delivery in a mousemodel of pancreatic cancer. Gut 2012.

51. Provenzano, P. P.; Cuevas, C.; Chang, A. E.; Goel, V. K.; Von Hoff,D. D.; Hingorani, S. R., Enzymatic Targeting of the Stroma AblatesPhysical Barriers to Treatment of Pancreatic Ductal Adenocarcinoma.Cancer Cell 2012, 21, (3), 418-429.

52. http://www.halozyme.com/files/2011EORTCPEGPH20.pdf.

53.http://ir.celgene.com/phoenix.zhtml?c=111960&p=irol-newsArticle&ID=1776848&highlight=.

54. Von Hoff, D. D.; Ramanathan, R. K.; Borad, M. J.; Laheru, D. A.;Smith, L. S.; Wood, T. E.; Korn, R. L.; Desai, N.; Trieu, V.; Iglesias,J. L.; Zhang, H.; Soon-Shiong, P.; Shi, T.; Rajeshkumar, N. V.; Maitra,A.; Hidalgo, M., Gemcitabine Plus nab-Paclitaxel Is an Active Regimen inPatients With Advanced Pancreatic Cancer: A Phase I/II Trial. Journal ofClinical Oncology 2011, 29, (34), 4548-4554.

55. Darland, D. C.; D'Amore, P. A., TGF-beta is required for theformation of capillary-like structures in three-dimensional coculturesof 10T1/2 and endothelial cells. Angiogenesis 2001, 4, (1), 11-20.

56. Quatromoni, J.; Wang, Y.; Vo, D.; Morris, L.; Jazirehi, A.; McBride,W.; Chatila, T.; Koya, R.; Economou, J., T cell receptor(TCR)-transgenic CD8 lymphocytes rendered insensitive to transforminggrowth factor beta (TGFbeta) signaling mediate superior tumor regressionin an animal model of adoptive cell therapy. Journal of TranslationalMedicine 2012, 10, (1), 127.

57. Zhang, Q.; Yang, X.; Pins, M.; Javonovic, B.; Kuzel, T.; Kim, S.-J.;Parijs, L. V.; Greenberg, N. M.; Liu, V.; Guo, Y.; Lee, C., AdoptiveTransfer of Tumor-Reactive Transforming Growth Factor-beta InsensitiveCD8+T Cells: Eradication of Autologous Mouse Prostate Cancer. CancerResearch 2005, 65, (5), 1761-1769.

B. Electron Microscopy Image of Tumor Tissue of Mice Injected withTGFβi-MSNP

An electron microscopic image of a BxPC3 tumor section from an animalinjected i.v. with TGFβi-MSNP is shown in FIG. 9.

C. Design, Synthesis, Drug Loading and Characterization ofGemcitabine-Loaded Liposomes Materials:

All the phospholipids products were purchased from Avanti Polar Lipids,either powder form or chloroform solution without further purification.Gemcitabine (GEM) was purchased from Sigma Aldrich. The liposomalmini-extruder, holder/heating block, and different size PC Membranes(0.4 and 0.1 μm) were purchased from Avanti Polar Lipids.

Optimization of GEM Loading in the Liposome Platform by Creating aTrans-Liposomal-Membrane Ammonium Sulfate Gradient.

The liposomal composition is shown in Table 1. Liposomes were preparedthrough a thin film-rehydration procedure. GEM was encapsulated using anequilibrium exchange method for liposomal trapping (BMC Cancer 2004, 4,63). Inclusion of ammonium sulfate inside the liposome generates atransmembrane gradient, which is responsible for protonation of theamphipathic GEM molecules which can freely diffuse through the liposomebilayer. However, after their protonation the GEM molecules becomehydrophilic, which prevents their escape from the liposome. The drugbecomes stabilized as gel-like drug precipitate [i.e. (GEM-NH3)2504]inside the liposomes. FIG. 10A provides a flow chart showing the majorsteps of GEM loading, and in order to obtain optimal drug loading, eachstep had to be systematically investigated through to find the bestpossible liposome formulation, ammonium sulfate concentration, extent ofsalt removal, drug loading time, temperature, and amount of free GEM,etc (FIG. 10B).

TABLE 1 Formulation of different liposome for GEM loading. Formulation#1 DOPC #2 DPPC #3 DOPC:Cholesterol:DSPE-PEG2K = 7:2:1 #4DOPC:Cholesterol:DSPE-PEG2K = 6:3:1 #5 DPPC:Cholesterol:DSPE-PEG2K =7:2:1 #6 DPPC:Cholesterol:DSPE-PEG2K = 6:3:1

Briefly, the lipid mixture for each formulation (#1-#6) was dissolved ina round-bottomed flask, using chloroform as solvent (concentration:2.5˜10 mg/mL). Different liposomal compositions are listed in Table 1.Based on the preliminary tests in which liposome size and polydispersityindices were compared, a decision was made to use formulation #5(DPPC:Cholesterol:DSPE-PEG2K=7:2:1) to perform further optimization ofloading because of the homogeneity of the liposome. Then lipid filmswere made by evaporation for ˜1 h, using a rotary evaporator connectedto a vacuum system at room temperature. These films were placed in achemical hood overnight to remove trace amounts of organic solventimpurities. The lipid films can be stored at −80° C. under inertatmosphere (i.e. argon, nitrogen) for at least 2 months. We alsoproduced fluorescent liposomes by co-dissolving 0.1% w/wfluorescein-DHPE (i.e. texas red) with the lipids. For rehydration,lipid films were incubated with indicated concentrations of ammoniumsulfate solution (ranging from 0-360 mM) at 60° C. for 1 h, withvigorous stirring. In order to make homogeneous unilamellar liposomes,the multi-lamellar particles were repeatedly extruded, first at a 400 nmpore size (3 times), then at a 100 nm pore size 11 times, while beingkept at 60° C. on a heating block. In order to remove the non-trappedammonium sulfate, ultra-speed centrifugation at 100,000 rpm or repeateddialysis against isotonic glucose solution was performed. The resultingmono-disperse, unilamellar vesicles were suspended in an isotonicsolution of GEM hydrochloride at different free GEM concentrations(0.2-5 mg/mL). These suspensions were kept at different temperatures(4-80° C.) for 1-24 and the free drug was removed by dialysis. In orderto determine the drug loading capacity, the encapsulated GEM wasquantified by UV absorption at 270 nm using a microplate reader(Molecular Device). The elemental phosphorus (P) was quantified byICP-OES (ICPE-9000, Shimadzu). Drug loading capacity (%) was determinedas (amount of GEM)/(amount of liposome)×100%. The size of the drug-ladenliposomes was characterized by dynamic light scattering at a liposomeconcentration of 100 μg/mL (ZetaPALS,

Brookhaven Instruments Co.). The zeta potential of the liposome wasmeasured by a ZetaPALS (Brookhaven Instruments Co.). The morphology ofdrug-laden liposome was visualized by cyroEM (TF20, FET).

After systematic variation of all the parameters, optimal GEMencapsulation could be achieved with:

A salt concentration: 120 nM

Dialysis: 3 dialysis cycles (6 mL against 1000 mL, 6 h/cycle)

Free GEM concentration: 1 mg/mL

Incubation temperature: 60° C.

Incubation time: 10 h

Through the use of optimal parameters, we could achieve a GEM loadingcapacity of ˜20% (w/w). Use of the optimal set of parameters (outlinedin green in FIG. 10A) allowed us to construct liposomes with a primaryparticle size of ˜137 nm, as shown by cyroEM image (FIG. 10C). Thehydrodynamic size in saline was 137 nm, with a potential of ˜1.7 mV(FIG. 10D). Stability testing demonstrated that GEM is stably retainedfor at least 21 days at 4° C., with <5.9% premature release.

Subsequent performance of confocal microscopy utilizing redfluorescent-labeled liposomes at 25 μg/mL, demonstrated a high rate ofcellular uptake in the human pancreatic cancer cell line, BxPC3, after a6 h incubation period (FIG. 10E, left panel). To demonstrate the invitro killing capability of the drug-laden liposomes, we used the MTSassay to compare the rate of BxPC3 cell death with incremental GEMconcentrations (FIG. 10E, right panel). Free GEM at the equivalent freedrug concentration was included as control. GEM delivery by the liposomeclearly linked to an increased rate of cytotoxicity compared to the freedrug. Liposomal drug encapsulation improves the IC50 ˜5 fold compared tofree drug at 48 h.

We also assess the ability of the liposome to protect GEM against theeffect of on cytidine deaminase (CDA), which plays a key role in druginactivation (circulatory half-life of <8 minutes) in the clinic.Briefly, free GEM or GEM-laden liposomes were mixed with CDA enzyme at aconcentration of 100 ng/mL in the incubation medium for 1 h at 37° C.The incubation media were then added to BxPC3 cells, which were culturedin 96 wells plate for 72 h. IC50 values were determined by the MTS assayfor each group. The results were compared to the IC50 values for GEM orGEM-liposomes, not incubated with CDA. Noteworthy, the effective invitro killing of the GEM-liposome was accompanied by protection of thedrug against CDA (FIG. 10F). By contrast, free GEM was not protectedagainst the effect of CDA.

D. Full Panel Of NIR Images to Cover all the Time Points in MiceInjected with Second Wave NIR-MNSP as Shown in FIG. 5A are shown in FIG.10 E. Demonstgration of The Effects of TGFβi-MSNP Treatment on Improvingthe Intratumoral Distribution of a “Hard Particle” in BxPC3 Xenografts

In order further confirm the effect of TGFβi-MSNP treatment on improvingbiodistribution of “hard particles”, 50 nm amine-modified, PEGylatedMSNP with a Dylight680 NIR tag were tested in xenograft tumors. The sameexperiment was performed as described in FIG. 5A. Briefly, the BxPC3-luctumor-bearing animals were pre-treated by i.v. injection of TGFβi-MSNP(inhibitor: 1 mg/kg; particle: 2 mg/kg) followed by i.v. injection of 50mg/kg of the 50 nm MSNP after a time lag of 1-2 h.

The in vivo biodistribution was compared with the mice receiving i.v.injection of the same amount of the 2nd wave particle alone. Consistentwith the results in FIG. 5, the MR fluorescent images showed that,compared to the treatment using the 2nd wave treatment alone, there wasa significant increase at tumor site in the mice prior treated withTGFβi-MSNP (FIG. 12).

F. Full Panel Of NIR Images to Show the Biodistribution of Second WaveNIR-Liposomes at all Time Points for the Experiment Described in FIG. 5Bis shown in FIG. 11.

TABLE 2 Table 2: Blood was collected from the sacrificed animals and thesera separated by centrifuging the whole blood at 5,000 rpm for 15 min.The biochemical parameters were assayed by the UCLA Division ofLaboratory Animal Medicine (DLAM) diagnostic laboratory services. Theseparameters include bicarbonate (CO2), cholesterol (CHOL), inorganicphosphorus (PHOS), aspartate aminotransferase (AST), direct serumbilirubin (DBILI), total bilirubin (TBILI), blood urea nitrogen (BUN),creatine kinase (CK), creatinine (CREAT), gamma glutamyl transferase(GGT), glucose (GLU), total protein (TP), albumin (ALB), alkalinephosphatase (ALP), calcium (CA), alanine aminotransferase (ALT), andBUN-to-creatinine ratio (BUN-CR). The biochemical analysis did not showany significant changes among different treatments. Parameters FreeTGFβi-MSNP groups Saline Liposome alone Free GEM GEM-Lip Two waves CO2(mEq/L) 24.4 ± 0.6  26.8 ± 1.3  23.2 ± 1.5  20.5 ± 2.8  21.4 ± 0.6  21.8± 2.7  CHOL (mg/dL) 78.0 ± 15.1 65.0 ± 2.6  73.0 ± 2   58.7 ± 4.0  43.0± 31.8 68.3 ± 11.8 PHOS (U/L) 5.4 ± 0.7 5.7 ± 0.9 4.8 ± 0.8 6.1 ± 1.06.2 ± 0.8 3.8 ± 0.7 AST (mg/dL) 156.9 ± 138.9 164.7 ± 66.0  150.9 ±79.3  221.8 ± 177.7 111.7 ± 93.7  275.4 ± 59.2  DBILI (mg/dL) 0.5 ± 0.30.5 ± 0.1 0.5 ± 0.2 0.7 ± 0.5 0.7 ± 0.2 0.6 ± 0.2 TBILI (mg/dL) 0.6 ±0.2 0.4 ± 0.1 0.4 ± 0.1 0.5 ± 0.2 0.4 ± 0.1 0.5 ± 0.2 BUN (mg/dL) 25.0 ±1.7  26.7 ± 3.0  33.7 ± 3.5  38.0 ± 11.3 32.7 ± 9.5  27.5 ± 2.3  CK(U/L) 362.5 ± 187.4 550.0 ± 134.5 647.0 ± 489.2 512.0 ± 39.6  550.0 ±84.8  743.0 ± 165.1 CREAT (mg/dL) 0.1 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.2 ±0.0 0.1 ± 0.1 0.2 ± 0.1 GGT (U/L) 5.7 ± 0.5 5.0 ± 1   4.7 ± 1.2  5.3 ±1.155  6.7 ± 0.577  6.5 ± 0.577 GLU (mg/dL) 101.0 ± 14.8  129.7 ± 81.4 174.0 ± 36.5  128.3 ± 16.9  129.0 ± 32.6  153.3 ± 12.8  TP (g/dL) 4.8 ±0.2 5.0 ± 0.2 5.1 ± 0.5 5.1 ± 0.2 5.3 ± 0.6 5.8 ± 0.4 ALB (g/dL) 2.9 ±0.1 3.0 ± 0.2 3.0 ± 0.2 3.2 ± 0.1 3.2 ± 0.2 3.5 ± 0.1 ALP (U/L) 29.3 ±13.6 43.9 ± 9.9  47.6 ± 8.2  55.8 ± 9.8  51.9 ± 27.2 77.3 ± 8.1  CA(mg/dL) 9.5 ± 0.2 9.9 ± 0.4 8.8 ± 1.3 9.4 ± 0.3 9.2 ± 0.5 9.7 ± 0.4 ALT(U/L) 19.6 ± 11.8 34.0 ± 7.5  25.9 ± 6.8  42.5 ± 33.8 26.5 ± 3.3  44.0 ±18.5 BUN_CR (mg/dL) 466.7 ± 378.6 190.1 ± 68.0  178.9 ± 63.8  190.7 ±16.0  281.9 ± 164.2 142.5 ± 45.4 

H. Materials and Methods Materials.

N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane (NAPTS) was purchasedfrom Gelest (Morrisville, Pa.). Cetyl trimethylammonium bromide (CTAB,95%), tetraorthoethylsilicate (TEOS, 98%), 3-(trihydroxysilyl) propylmethylphosphonate (42% in H₂O), Pluronic F127, polyethyleneimine (PEI,1.2 kD), 4-(dimethylamino)pyridine (99%), N,N′-disuccinimidyl carbonate(95%), poly(ethylene glycol) methyl ether (m-PEG, MW 5 kD), phthalicanhydride (99%), transforming growth factor-β1 (TGF-β) and gemcitabinehydrochloride (purity: ≧98%) were purchased from Sigma Aldrich (St.Louis, Mo.) Amine-reactive near-infrared Fluor Dylight 680 NHS ester waspurchased from Thermo Scientific (Rockford, Ill.). D-Luciferin waspurchased from Xenogen (Alameda, Calif.). Cell Tracker™ Red CMTPX, CellTracker™ Green CMFDA (5-Chloromethylfluorescein Diacetate), DPBSsolution, L-glutamine, penicillin, streptomycin, and DMEM culture mediumwere obtained from Invitrogen. Fetal bovine serum (FBS) was purchasedfrom Atlanta Biologicals.

Anti-Smad2 (phospho S467) antibody was purchased from Abcam. Anti-CD31antibody and Matrigel™-Basement Membrane Matrix was purchased from BDBioscience. Transforming growth factor type I receptors kinas inhibitor(TGFβi, LY364947) was purchased from EMD Millipore. Phospholipids andcholesterol were purchased from Avanti Polar Lipids (Alabaster, Ala.).All reagents were used without further purification.

Physicochemical Characterization.

Samples were characterized for morphology, size distribution and surfacecharge. The morphologies and primary sizes of MSNP particle werecharacterized using a transmission electron microscope (JEOL JEM 2010,JEOL USA, Inc., Peabody, Mass.). The morphologies of liposome werecharacterized using cyroEM (TF20, FET). Hydrodynamic size and zetapotential in solution were measured by ZetaSizer Nano (MalvernInstruments Ltd., Worcestershire, UK). All of the measurements wereperformed with the samples suspended in filtered water or saline at 100μg/mL nanoparticle concentration.

Establishment of BxPC3-luc cells.

Permanent luciferase transfection using letivirus was performed by UCLAvector core facility. Briefly, 1.5×10⁴ BxPC3 cells immersed in 40 μLcomplete DMEM were transduced with 10 μL of lentivirus solution (CignalFinder Lenti Pathway Reporter Qiagen/SA Biosciences; 1.4×10⁷ TU/mL)using 96 well tissue culture plates. Centrifugal inoculation wasperformed at 1,200 g for 60 minutes. The viral containing media wasremoved after 16 h and the cultures replenished with fresh DMEM media.Cells were allowed to proliferate to a population size of 1.2×10⁶ cells.Limiting dilution was used to select individual cell that express thehighest luciferase. The highest luciferase expressing clone (refers asBxPC3-luc) out of 10 single clones was used for further experiments.

ICP-OES Analysis:

The collected tumor and organs were used for Si elemental analysis usingICP-OES. Briefly, each tissue was accurately weighed and soaked inconcentrated 1 mL HNO₃ and 0.5 mL 30% H₂O₂ for overnight. This yellowcolor digestion solution was heated at 80° C. for 1 h in the subsequentday. Dropwise addition of H₂O₂ solution was used to drive off nitrogenoxide vapor until the digestion lipid turns colorless. 2% HNO₃ was usedto dilute the sample into 10 mL volume and the resulting sample wasanalyzed by ICP-OES.

Blood Biochemistry and Histology to Assess Possible Toxicity

Following the animal experiments described above, the mice weresacrificed on the 38^(th) day and serum was collected by centrifugingthe whole blood at 5,000 rpm for 15 min. The biochemical parameters wereassayed by UCLA Division of Laboratory Animal Medicine (DLAM) diagnosticlaboratory services. Appropriate size sections of the tumor, liver,kidney, and spleen were fixed in 10% formalin and then embedded intoparaffin. Tissue sections of 4 μm thickness were mounted on glass slidesby the UCLA Division of Laboratory Animal Medicine (DLAM) diagnosticlaboratory services. The sections were stained with hematoxylin-eosin(H&E) and examined by light microscopy.

Example II A. Introduction

An MSNP coated with a phospholipid bilayer is described which canprovide a GEM loading capacity of ˜40% (drug/particle, w/w). The MSNPcore is synthesized by a modified surfactant-templated sol-gel method inaqueous solution at relatively low temperature. Moreover, to coat thisNP with an intact lipid bilayer, a lipid membrane was dehydrated with aGEM-containing MSNP suspension, using controlled energy input (e.g.sonication). This led to rapid coating and sealing of the MSNPs,encapsulating a high w/w content GEM in one step.

Since pancreatic cancer can in many cases be resistant to individualchemotherapeutic agents, including GEM, via acquired or de novomechanisms, there is a need to consider drugs that provide a synergisticeffect with GEM, e.g., when co-administered with GEM. In this regard, arecent successful clinical trial has allowed Abraxane^(R)(paclitaxel/albumin complex) to be combined with GEM in untreatedpancreatic patients with metastatic disease. This combination hasresulted in a statistically significant improvement in overall survivalcompared to patients receiving GEM alone. Without wishing to be bound bytheory, paclitaxel is believed to be capable of increasing theintratumoral GEM content by reducing the activity of cytidine deaminase(CDA), a key enzyme that metabolically inactivates GEM and reduces itscirculatory half-life to minutes. Since the hydrophobic paclitaxelmolecules can be co-dissolved in a lipophilic organic solution, thepresence of a lipid coat on MSNP allows co-packaging of paclitaxel in aphospholipid bilayer coating on GEM-laden particles.

Reports by Celano et al. in 2004 and Cosco et al. in 2009 discuss usinga liposome for GEM delivery (see BMC Cancer 2004, 4:63; Cancer ChemotherPharmacol, 2009, 64:1009-1020, each of which is incorporated byreference in its entirety). In the 2004 study, the authors describe aGEM loading capacity 0.3% (w/w, drug/particle). In the 2009 year study,the authors describe GEM loading capacity of 1.3% (w/w, drug/particle).Reports by Brinker et al. discuss porous silica nanoparticle-supportedlipid bilayers, also known as “protocells” for drug delivery (Nat.Materials 2011, 10, 389, which is incorporated by reference in itsentirety).

In some embodiments, a low temperature sol-gel chemistry procedure isused to obtain highly uniform (e.g., monodisperse) and colloidallystable MSNPs. MSNPs prepared via a low temperature sol-gel method canexhibit improved size control than those prepared by, for example, anaerosol-assisted self-assembly method. Particles prepared by anaerosol-assisted self-assembly method may exhibit a wide sizedistribution, and may not be uniformly bio-available, e.g., at tumorsites. In contrast, the monodisperse and size-controlled MSNPs preparedby a sol-gel method may show greater potential for in vivo use.

In some embodiments, a submicron structure (such as an MSNP coated witha phospholipid bilayer) can provide simultaneous delivery of a drug,and: an agent which stabilizes the drug against metabolic degradation;an agent which facilitates the delivery of the drug to a target cell,tissue, organ or tumor; an agent which acts synergistically with thedrug; one or more additional therapeutic agents; or a combinationthereof In some embodiments, a GEM-laden MSNP provides for simultaneousdelivery of paclitaxel in a single carrier, i.e., a submicron structurewhich includes both GEM (e.g., within the pores) and paclitaxel (e.g.,associated with the phospholipid bilayer). Including more than onetherapeutic agent in a single particle allows precise control over thedoses and dosage ratios of the therapeutic agents delivered to the siteof release (e.g., a tumor cell).

B. Synthesis Procedure of Lipid-Coated MSNP and Drug Loading B1.Synthesis of MSNP Via Sol-Gel Chemistry Chemicals:

The chemicals were obtained from Sigma Aldrich and used without furtherpurification.

Small Batch MSNP Synthesis:

5 mL cetyltrimethylammonium chloride (CTAC) (25%) was mixed with 15 mLH₂O, and stirred for 15 min at 75° C. (350 rpm). Added 0.8 mL 10% TEAwater sollution, mixed at 75° C. for 15 min (350 rpm). Added dropwise 1mL tetraethyl orthosilicate (TEOS) as silica precursor, at a rate of 30drops per minute. The mixture was stirred continuously at 350 rpm at 80°C. for 1 h. A white nanoparticle suspension gradually developed, with aprimary size of about 60 nm to about 70 nm.Scaled up MSNP synthesis:25 mL CTAC (25%) was mixed with 75 mL H₂O, at 95° C. in a 200 mL conicalflask. 4 mL 10% TEA, was added, the mixture was stirred at 95° C. for 30min. A pump was used to deliver 7.5 mL TEOS at 1 ml/min to the flask.The reaction was allowed to proceed at 95° C. for 20 min. TEM analysisshowed the primary particle size of ˜70 nm.B2. Washing to remove surfactantPrepared washing buffer containing methanol and HCl at 500:19 (v/v).Added 50 mL acidic washing buffer into scaled up synthesis system (˜100mL). Stirred at 350 rpm at room temperature for overnight. Spun downparticles at 15,000 rpm for 10 min (1.5 mL tube) or 10,000 rpm for 30min (50 mL tube). Used probe-sonicator to re-suspend the particles usingfresh methanol Washed the particles for at least 3 times. Frequent DLSanalysis was carried out to confirm the absence of particleaggregation/contamination. TEM was used to confirm the particlemorphology before use. IR or cytotoxicity assay was used to confirmwhether the detergent was thoroughly removed.

B3. Drug Loading

10 mg MSNP was suspended in 20 mg GEM ethanol/water (7:3, v/v). Themixture was shaken for at least 24 hour at room temperature. Thedrug-laden particles were collected by centrifugation (prior to poresealing) and immediately used for lipid coating. Particles were notwashed between drug loading and lipid coating.

B4. Phospholipid Bilalyer Coating of GEM-Laden MSNP

The lipid membrane was dehydrated using GEM-containing MSNP suspensionwith controlled energy input (e.g. sonication), leading to lipid-coatedand pore-sealed MSNPs that contained high GEM content in one step. Lipidmembrane: Lipid mixture was dissolved in a round-bottomed flask, usingchloroform as solvent (concentration: 2.5˜10 mg/mL). Different liposomalcompositions can be selected based on drug, targeting purpose, and otherconsiderations. Paclitaxel can be co-dissolved in the organic solution.Lipid films were made by evaporation for ˜1 h, using a rotary evaporatorconnected to a vacuum system at room temperature. These films wereplaced in a chemical hood for at least 2 hours to remove trace amountsof organic solvent impurities.

The lipid films can be stored at −80° C. under an inert atmosphere forat least 2 months. Fluorescently labeled lipid film can be made byco-dissolving 0.1% w/w fluorescein-DHPE (i.e. Texas red) with thelipids. For rehydration, lipid films were incubated with the GEM-ladenMSNP aqueous solution at 40° C. for 20 min, with continuous water-bathsonication. The mixture was spun at 1500 rpm for 5 min and thesupernatant collected, which contain lipid-coated MSNP, free GEM, andfree liposome. A centrifugal filter unit with 10,000 MW cutting off sizewas used to remove any un-encapsulated GEM.

Sample characterization: The samples were fully characterized formorphology using TEM and cryoEM, size and zeta potential, surface area,loading capacity and release profile, and Si/P elemental ratio usingICP-OES.

C. In vitro demonstration to show the effects of GEM/paclitaxel loadedlipid-coated MSNP in pancreatic cancer cells.

C1. Determination of Morphology and GEM Loading Capacity in MSNP

CryoEM images of lipid coated MSNP are shown in FIG. 14, which shows acryoEM image (TF20, FET) of lipid-coated MSNP. The upper box shows the˜70 nm MSNP synthesized using the procedure in section D. The zoom-inimage (region 1) shows an ordered mesoporous structure and primaryparticle size of ˜60 nm. The lower panel shows an intact lipid coatingon the silica surface. The zoom-in images (regions of ii→iv) showed alipid thickness of 7.0 nm on the silica surface, which is very close tothe thickness of lipid bilayer in liposome (7.1 nm). The HPLC analysisand microplate reader analysis showed that the loading capacity of GEMin lipid coated MSNP was ˜40% (w/w). This is an approximately 2-foldimprovement compared to GEM-laden liposome.

C2. Cellular Uptake of the Lipid Bilayer-Coated MSNP and its Ability toProvide Intracellular Delivery of Cancer Drugs in Panc1 Cells

FIG. 15 demonstrates cellular uptake of the lipid bilayer-coated MSNP.Confocal microscopy was used to demonstrate the cellular uptake ofFITC-labeled paclitaxel (in green) loaded DHPE (red)-labeledlipid-coated MSNP in Panc1 cells. Panc1 cells were treated with 40 μg/mLnanoparticles for the indicated time periods. The merged image at 3hours showed the red-labeled lipid bilayer in association withgreen-labeled paclitaxel, which indicated that the particle successfullydelivered paclitaxel into the cells, while the lipid coating remainedintact. However, the merged images at 20 hours show amore dispersedpattern of intracellular paclitaxel distribution and lower level ofco-localization, providing evidence of intracellular paclitaxel releasefrom the MSNP's lipid coating where the hydrophobic drug was packaged.The nuclear were stained by Hoechst 33342 in blue.

C3. Paclitaxel-Laden Lipid Coated MSNP Down-Regulated the Expression ofCytidine Deaminase (CDA), a Key Enzyme in Metabolic Inactivation of GEMin Pancreatic Cancer Cells.

As illustrated in FIG. 16, Panc1 cells were treated withpaclitaxel-laden lipid coated MSNP at 25 μg/mL for different lengths oftime (0-24 hours), or for 24 hours using different particleconcentrations (0-200 μg/mL). The expression of CDA and heme oxygenase-1(H0-1, which is an oxidative stress protein induced in response topaclitaxel induced oxidative challenge) were determined by Westernblotting. The data demonstrated that paclitaxel-laden lipid coated MSNPsignificantly lowered the CDA expression and induced HO-1 level in adose- and time-dependent manner. The data showed that 24-hour incubationat particle concentration of 25 μg/mL could lead to the maximal effectsin Panc1 cells.

1-29. (canceled)
 30. A method of making a liposome suitable to receive atherapeutic agent therein, the method comprising: a) preparing a lipidfilm; and b) hydrating the lipid film in the presence of a protonatingagent to create a liposome with a protonating agent therein, wherein theliposome comprises a particle comprising a silica body having aplurality of pores and having a maximum dimension of less than onemicron, the silica body having a surface coated with a phospholipidbilayer; and the phospholipid bilayer covering the plurality of poresand encapsulating the particle.
 31. The method of claim 30, furthercomprising: c) incubating the liposome with a therapeutic agent.
 32. Themethod of claim 31, wherein the therapeutic agent diffuses through thephospholipid bilayer and is protonated.
 33. The method of claim 32,wherein the resulting liposome comprises at least about 10% w/w or atleast about 20% w/w of the therapeutic agent.
 34. The method of claim33, wherein the therapeutic agent is gemcitabine.
 35. The method ofclaim 34, wherein the gemcitabine is disposed within the resultingliposome in the form of a gel-like precipitate.
 36. The method of claim35, wherein the phospholipid bilayer is loaded with a hydrophobicmolecule.
 37. The method of claim 36, wherein the hydrophobic moleculeis paclitaxel.
 38. The method of claim 30, wherein the protonating agentis selected for its ability to react in a protonation reaction andthereby convert an amphiphatic molecule into a hydrophilic molecule. 39.A liposome made by the method of claim
 35. 40. A submicron particleincluding a silica body having a plurality of pores suitable to receivea therapeutic agent therein, and having a surface, and a phospholipidbilayer coating the surface, wherein the submicron particle has amaximum dimension of less than one micron, and the phospholipid bilayerstably seals the plurality of pores and encapsulates the particle; andthe submicron particle includes about 10% w/w or greater of therapeuticagent within the pores.
 41. The submicron particle of claim 40, whereinthe submicron particle includes about 20% w/w or greater of moleculeswithin the pores.
 42. The submicron particle of claim 41, wherein thetherapeutic agent is gemcitabine.
 43. The submicron particle of claim42, wherein the gemcitabine is disposed in the pores in the form of agel like precipitate.
 44. The submicron particle of claim 43, whereinthe particle comprises paclitaxel disposed within the lipid bilayer. 45.The submicron particle of claim 40, wherein the particle comprisesammonium sulfate disposed therein.
 46. A method for inhibiting thegrowth of a human pancreatic ductal adenocarcinoma (HPDAC) cellcomprising: (a) combining the HPDAC cell with a submicron particleincluding a silica body having a plurality of pores suitable to receivemolecules therein, and having a surface, and a phospholipid bilayercoating the surface, wherein the submicron particle has a maximumdimension of less than one micron, and the phospholipid bilayer stablyseals the plurality of pores and encapsulates the particle; and thesubmicron particle includes about 10% w/w or greater of gemcitabinewithin the pores, wherein the gemcitabine is disposed in the pores inthe form of a gel like precipitate; and (b) allowing the gemcitabine tomigrate from the pores of the submicron particle into the HPDAC cellsuch that the growth of the HPDAC cell is inhibited.
 47. The method ofclaim 46, wherein the submicron particle includes about 20% w/w orgreater of gemcitabine within the pores.
 48. The method of claim 47,wherein the submicron particle comprises paclitaxel disposed within thelipid bilayer.
 49. The method of claim 48, wherein the HPDAC cell iscombined with the submicron particle in vivo.